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Abstract:

Anti CMV antibodies are provided. Thus an antibody of the present
invention comprises an antigen recognition domain capable of binding an
MHC molecule being complexed with a cytomegalovirus (CMV) pp65 or pp64
peptide, wherein the antibody does not bind said MHC molecule in an
absence of said complexed peptide, and wherein the antibody does not bind
said peptide in an absence of said MHC molecule. Also provided are
methods of using the antibodies.

Claims:

1. An antibody comprising an antigen recognition domain capable of
binding an MHC molecule being complexed with a cytomegalovirus (CMV) pp65
or pp64 peptide as set forth by SEQ ID NO:3, wherein the antibody does
not bind said MHC molecule in an absence of said complexed peptide, and
wherein the antibody does not bind said peptide in an absence of said MHC
molecule.

2. The antibody of claim 1, being conjugated to a therapeutic moiety.

3. The antibody of claim 1, attached to a detectable moiety.

4. The antibody of claim 1, being an antibody fragment.

5. An antibody comprising a multivalent form of the antibody of claim 1.

6. The antibody of claim 5, wherein said multivalent form is an IgG
antibody.

7. A pharmaceutical composition comprising as an active ingredient the
antibody of claim 1.

8. A method of detecting a cell expressing a cytomegalovirus (CMV)
antigen, comprising contacting the cell with the antibody of claim 1
under conditions which allow immunocomplex formation, wherein a presence
or a level above a predetermined threshold of said immunocomplex is
indicative of CMV expression in the cell.

9. A method of diagnosing a cytomegalovirus (CMV) infection in a subject
in need thereof, comprising contacting a cell of the subject with the
antibody of claim 1 under conditions which allow immunocomplex formation,
wherein a presence or a level above a pre-determined threshold of said
immunocomplex in the cell is indicative of the CMV infection in the
subject.

10. A method of treating a disease associated with cytomegalovirus (CMV)
infection, comprising administering to a subject in need thereof a
therapeutically effective amount of the antibody of claim 1, thereby
treating the disease associated with CMV infection.

11. The method of claim 9, wherein said subject has a suppressed or a
compromised immune system.

12. The method of claim 9, wherein said CMV infection is associated with
a disease selected from the group consisting of mononucleosis, retinitis,
pneumonia, gastrointestinal disorders, and encephalitis.

13. The method of claim 9, wherein said cell is a retina cell, lung
epithelial cell, a gastrointestinal epithelial cell or a brain cell.

14. The method of claim 9, wherein said subject is an immuno-compromised
organ transplant recipient.

15. The method of claim 9, wherein said subject is infected with human
immunodeficiency virus (HIV).

Description:

RELATED APPLICATIONS

[0001] This application is a division of U.S. patent application Ser. No.
12/450,476 filed on Jan. 6, 2010, which is a National Phase of PCT Patent
Application No. PCT/IL2008/000437 having International filing date of
Mar. 27, 2008, which claims the benefit of priority of U.S. Provisional
Patent Application Nos. 60/929,207 filed on Jun. 18, 2007 and 60/907,343
filed on Mar. 29, 2007. The contents of the above applications are all
incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

[0002] The present invention, in some embodiments thereof, relates to
methods of diagnosing and treating cytomegalovirus diseases and, more
particularly, but not exclusively, to antibodies capable of same.

[0003] Of all the human herpesviruses described to date, infection with
cytomegalovirus (CMV) is considered to be the main cause of morbidity and
mortality. Approximately 70% of the world population are carriers of the
virus. Primary infection with the virus results in a life long
persistence in a latent form and is therefore generally asymptomatic in
healthy adults. However, some individuals, such as immuno-compromised
organ transplant recipients, or individuals infected with human
immunodeficiency virus (HIV), are at high risk of developing life
threatening CMV disease due to CMV reactivation. In addition, CMV has
emerged in recent years as the most important cause of congenital
infection in the developed world, commonly leading to mental retardation
and developmental disability.

[0004] Immunity to CMV is complex and involves humoral and cell-mediated
responses. Studies showed that both natural killer (NK) cells and
cytotoxic T-lymphocytes (CTLs) are of primary importance in prevention of
recurrence. Many gene products participate in generating the CTL response
to CMV infection, however, the high level expression frequencies of the
viral protein pp65 (e.g., Genbank Accession No. M15120; SEQ ID NO:48)
suggests pp65 as the main target of the CTL-mediated immune response.
Among all pp65 peptides, CMV specific-CTL activity in HLA-A2 positive
individuals was found to be mainly directed to the peptide
pp65495-503 (NLVPMVATV; SEQ ID NO:3) (Chee M S et al., 1990).

[0005] Cytosolic proteins, usually synthesized in the cells, such as CMV
viral proteins, enter the class I MHC pathway of antigen presentation. In
the first step, ubiquitinated cytoplasmic proteins are degraded by the
proteasome, a cytoplasmic multiprotein complex which generates a large
portion of peptides destined for display by class I MHC molecules.
Peptides are then delivered from the cytoplasm to the endoplasmic
reticulum (ER) by the transporter associated with antigen presentation
(TAP) molecules. Newly formed class I MHC dimers in the ER associate with
and bind peptides delivered by the TAP. Peptide binding stabilizes class
I MHC molecules and permits their movement out of the ER, through the
Golgi apparatus, to the cell surface. This pathway ensures that any cell
synthesizing viral proteins can be marked for recognition and killing by
CD8+ CTL.

[0010] According to an aspect of some embodiments of the present invention
there is provided an antibody comprising an antigen recognition domain
capable of binding an MHC molecule being complexed with a cytomegalovirus
(CMV) pp65 or pp64 peptide, wherein the antibody does not bind the MHC
molecule in an absence of the complexed peptide, and wherein the antibody
does not bind the peptide in an absence of the MHC molecule.

[0011] According to an aspect of some embodiments of the present invention
there is provided an antibody comprising a multivalent form of the
antibody of the present invention.

[0012] According to an aspect of some embodiments of the present invention
there is provided a pharmaceutical composition comprising as an active
ingredient the antibody of the antibody of the present invention.

[0013] According to an aspect of some embodiments of the present invention
there is provided a method of detecting a cell expressing a
cytomegalovirus (CMV) antigen, comprising contacting the cell with the
antibody of the present invention under conditions which allow
immunocomplex formation, wherein a presence or a level above a
predetermined threshold of the immunocomplex is indicative of CMV
expression in the cell.

[0014] According to an aspect of some embodiments of the present invention
there is provided a method of diagnosing a cytomegalovirus (CMV)
infection in a subject in need thereof, comprising contacting a cell of
the subject with the antibody of the present invention under conditions
which allow immunocomplex formation, wherein a presence or a level above
a pre-determined threshold of the immunocomplex in the cell is indicative
of the CMV infection in the subject.

[0015] According to an aspect of some embodiments of the present invention
there is provided a method of treating a disease associated with
cytomegalovirus (CMV) infection, comprising administering to a subject in
need thereof a therapeutically effective amount of the antibody of the
present invention, thereby treating the disease associated with CMV
infection.

[0016] According to some embodiments of the invention, the cytomegalovirus
(CMV) pp65 or pp64 peptide is set forth by SEQ ID NO:3.

[0017] According to some embodiments of the invention, the antigen
recognition domain comprises complementarity determining region (CDR)
amino acid sequences as set forth in SEQ ID NOs:24-26 and 30-32.

[0018] According to some embodiments of the invention, the antigen
recognition domain comprises complementarity determining region (CDR)
amino acid sequences as set forth in SEQ ID NOs: 36-38 and 42-44.

[0019] According to some embodiments of the invention, the antibody being
conjugated to a therapeutic moiety.

[0020] According to some embodiments of the invention, the antibody is
attached to a detectable moiety.

[0021] According to some embodiments of the invention, the antibody being
an antibody fragment.

[0022] According to some embodiments of the invention, the multivalent
form is an IgG antibody.

[0023] According to some embodiments of the invention, the subject has a
suppressed or a compromised immune system.

[0024] According to some embodiments of the invention, the CMV infection
is associated with a disease selected from the group consisting of
mononucleosis, retinitis, pneumonia, gastrointestinal disorders, and
encephalitis.

[0025] According to some embodiments of the invention, the cell is a
retina cell, lung epithelial cell, a gastrointestinal epithelial cell or
a brain cell.

[0026] According to some embodiments of the invention, the subject is an
immuno-compromised organ transplant recipient.

[0027] According to some embodiments of the invention, the subject is
infected with human immunodeficiency virus (HIV).

[0028] Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Although methods and
materials similar or equivalent to those described herein can be used in
the practice or testing of the present invention, suitable methods and
materials are described below. In case of conflict, the patent
specification, including definitions, will control. In addition, the
materials, methods, and examples are illustrative only and not intended
to be limiting.

[0029] As used herein, the terms "comprising" and "including" or
grammatical variants thereof are to be taken as specifying the stated
features, integers, steps or components but do not preclude the addition
of one or more additional features, integers, steps, components or groups
thereof. This term encompasses the terms "consisting of" and "consisting
essentially of".

[0030] The phrase "consisting essentially of" or grammatical variants
thereof when used herein are to be taken as specifying the stated
features, integers, steps or components but do not preclude the addition
of one or more additional features, integers, steps, components or groups
thereof but only if the additional features, integers, steps, components
or groups thereof do not materially alter the basic and novel
characteristics of the claimed composition, device or method.

[0031] The term "method" refers to manners, means, techniques and
procedures for accomplishing a given task including, but not limited to,
those manners, means, techniques and procedures either known to, or
readily developed from known manners, means, techniques and procedures by
practitioners of the biotechnology and medical art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office upon
request and payment of the necessary fee.

[0033] Some embodiments of the invention are herein described, by way of
example only, with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of illustrative
discussion of embodiments of the invention. In this regard, the
description taken with the drawings makes apparent to those skilled in
the art how embodiments of the invention may be practiced.

[0036] FIGS. 2a-d are flow cytometry analyses depicting the detection of
MHC-peptide complexes on the surface of APCs using the H9 and F5 soluble
Fabs. JY or RMAS-HHD cell lines were pulsed with various specific and
nonspecific peptides. JY cells (FIGS. 2a and c) or RMAS-HHD cells (FIGS.
2b and d) loaded with the CMV pp65495-503 peptide (SEQ ID NO:3) or
control peptides ("280", "540"), incubated with the H9 (FIG. 2a, b) or F5
(FIGS. 2c, d) Fab respectively. Specific staining of the pp65 loaded
cells, but not the control cells, is shown. The same type of assay was
performed with 10 different control HLA-A2-restricted peptides (data not
shown).

[0037] FIGS. 3a-c are flow cytometry analyses depicting the detection of
MHC-peptide complexes on the surface of JY cells using H9Fab in its
monomeric or tetrameric forms. The JY cell line was pulsed with different
peptides. FIG. 3a--JY cells loaded with pp65495-503 peptide (SEQ ID
NO:3). Incubations were with H9Fab monomer and PE-labeled anti human Fab,
or with H9Fab tetramer connected to PE labeled streptavidin. FIG. 3b--JY
cells loaded with pp65495-503 peptide (SEQ ID NO:3). Incubations
were with H9Fab monomer and FITC-labeled anti human Fab, or with H9 whole
IgG Ab and FITC-labeled anti human Fab. FIG. 3c--JY cells loaded with
gp100280-288 280-288 (SEQ ID NO:4) as a control. Incubations were
with H9Fab monomer and PE-labeled anti human Fab, H9Fab tetramer
connected to PE labeled streptavidin or with H9 IgG Ab and PE-labeled
anti human Fab. Note the specific binding of the H9Fab in its monomeric
or tetrameric form, as well as the whole IgG H9 Ab to JY cells pulsed
with the HLA-A2-CMV peptide (pp65 495-503) but not with JY cells when
pulsed with the control peptide (gp100 280-288). Also note the increased
avidity of the IgG Ab as compared to the monomeric Fab, or the increased
avidity of the tetrameric Fab form as compared to the monomeric Fab form.

[0038] FIGS. 4a-c are graphs depicting the affinity determination of the
H9 Ab in its monomeric (FIG. 4a) or IgG (FIG. 4b) forms, as detected by
surface plasmon resonance (SPR) analysis. Each of the forms was flowed
over the relevant wells at three different concentrations (0.05 μM,
0.1 μM, 0.2 μM) of biotinylated HLA-A2-pp65 495-503 complexes. As a
control, H9 Ab were flowed over wells which were coated with control
biotinylated HLA-A2/pEBV complexes (FIG. 4c). Note the absence of binding
signal of the H9 Ab over the HLA-A2/pEBV complex (the concentration of
HLA-A2/pEBV complex was 0.2 μM) as compared to the HLA-A2/pp65 complex
(the concentration of HLA-A2/pp65 complex was 0.2 μM).

[0039] FIGS. 5a-c are flow cytometry analyses depicting the detection of
Fab sensitivity threshold (FIGS. 5a-b) and of rare cells bearing the
specific peptide-MHC complex in a heterogenous cell population (FIG. 5C).
In order to detect Fab sensitivity threshold, JY cells were pulsed with
various concentrations of pp65495-503 peptide (0.65 nM, 0.1 μM,
012 μM, 0.25 μM, 0.5 μM, 1 μM or 100 μM), and incubated
with H9Fab monomer (at a concentration of 10 μg/ml) and PE-labeled
anti human Fab (FIG. 5a), or H9 Fab tetramer (at a concentration of 10
μg/ml) connected to PE labeled streptavidin (FIG. 5b). Note the
significantly low concentration of the pp65495-503 peptide needed to
pulse JY cells in order to obtain a significant binding with the H9
tetramer [e.g., a threshold of 65 nM) of the pp65 495-503 peptide] or the
H9 monomer [e.g., a threshold of 0.1 μM of the pp65 495-503 peptide].
Detection of rare population of cells bearing the specific MHC-peptide
complex was by pulsing JY APCs with the pp65 495-503 peptide and mixing
them with APD cells (HLA-A2-B cell line) at different ratios (FIG. 5C) so
as to obtain pre-determined concentrations of cells expressing the
specific MHC-peptide complex. The mixed population was stained with H9Fab
(at a concentration of 10 μg/ml), and detection sensitivity was
monitored by flow cytometry. Note the specific detection of as low as 5%
cells bearing the specific MHC-pp65 495-503 complex.

[0040] FIGS. 6a-m are flow cytometry analyses (FIGS. 6a-1) and a bar graph
(FIG. 6m) depicting the detection of the specific HLA-A2/pp65 complex by
H9 tetramer (FIG. 6e-h) or H9 IgG Ab (Data not shown), after naturally
occurring active intracellular processing. HLA-A2 positive fibroblasts
were infected with the CMV laboratory strain AD169 (FIGS. 6a, e, i).
HLA-A2 positive uninfected fibroblasts were used as a control (FIGS. 6c,
g, k) as well as HLA-A2 negative infected fibroblasts (FIGS. 6b, f, j) or
HLA-A2 negative uninfected fibroblasts (FIGS. 6d, h, l). Incubation were
with PE labeled BB7.2 (FIGS. 6a-d), PE labeled H9 tetramer (FIGS. 6e-h)
or anti pp65 FITC mAB (FIGS. 6i-1) followed by the secondary antibody
FITC-labeled anti mouse IgG, 72 hours after infection. Note the specific
binding of the H9 tetramer to HLA-A2 positive cells following infection
with CMV (FIG. 6e) as compared to the absence of binding to HLA-A2
negative cells (FIG. 60 or to uninfected cells (FIGS. 6g and h),
demonstrating the specific HLA-A2-CMV (pp65 495-503) complex--dependent
binding of the H9 antibody to cells ex vivo. In contrast, note the
non-CMV-dependent binding of the BB7.2 Ab to HLA-A2 positive cells [same
binding efficacy in the presence (FIG. 6a) or absence (FIG. 6c) of CMV
peptide], and the non-HLA-A2-dependent binding of the Anti pp65 Ab in
CMV-infected cells [same binding efficacy in HLA-A2 positive (FIG. 6i) or
HLA-A2 negative (FIG. 6j) cells]. FIG. 6m--A cytotoxicity assay by which
H9 IgG Ab is shown to block virus infected cells killing mediated by
specific CTL line. Fibroblast cells were radioactively labeled with
S35 methionine before infection with the CMV virus and 72 hours
later the cells were incubated with the H9 IgG Ab. CTLs were added at a
target (fibroblast cells infected with CMV):effector (CTL) ratio of 1:10
and incubated for five hours. Cells incubated with W6 Ab (an antibody
directed against HLA-A,B,C) were used as positive control, while cells
without any Ab incubation served as a reference for the maximum killing
rate. These results demonstrate the TCR-like specificity of the H9 IgG Ab
to specific CMV-infected cells.

[0043] FIGS. 9a-y are flow cytometry analyses depicting kinetic assays
which follow the dynamics between the HLA-A2 extracellular presentation,
the HLA-A2/pp65 peptide extracellular and intracellular complex
presentation and the pp65 expression, in HLA-A2+ (positive) uninfected
cells (FIGS. 9a-t) or in HLA-A2-(negative) cells infected with the AD169
Wild Type strain of CMV. Staining with the H9 IgG antibody, BB7.2
antibody or the anti pp65 antibodies was effected in the uninfected cells
harvested at parallel times [i.e., 36 (FIGS. 9a-e), 72 (FIGS. 9f-j), 96
(FIGS. 9k-o), and 120 (FIGS. 9p-t) hours] to the cells infected with the
viruses as described in FIGS. 7a-t and 8a-t, hereinabove. Infected
HLA-A2-(negative) cells were harvested and stained with the H9 IgG
antibody, BB7.2 antibody or the anti pp65 antibody at 120 hours after
infection with the AD169 CMV virus. Extracellular staining with the H9
IgG antibody is shown in FIGS. 9a, f, k, p and u. Intracellular staining
with the H9 IgG antibody is shown in FIGS. 9b, g, l, q and v.
Extracellular staining with the BB7.2 antibody is shown in FIGS. 9c, h,
m, r and w. Intracellular staining with the BB7.2 antibody is shown in
FIGS. 9d, i, n, s and x. Staining with the anti pp65 antibody is shown in
FIGS. 9e, j, o, t, and y. FITC-labeled anti mouse antibody and Alexa
fluor488-labeled anti human antibody were used as secondary
antibodies for the anti pp65 mAb and the H9 IgG Ab respectively.
Intracellular staining was feasible by cells permeabilization.

[0044] FIGS. 10a-d are bar graphs depicting quantization of the number of
HLA-A2/pp65 complexes inside and on the surface of virus infected cells.
The level of fluorescence intensity on stained cells was compared with
the fluorescence intensities of calibration beads with known numbers of
PE molecules per bead, thus providing a mean of quantifying PE-stained
cells using a flow cytometer. Incubations were with BB7.2 PE labeled Ab
(FIGS. 10c and d), and H9 Ab (FIGS. 10a and b). PE-labeled anti kappa
antibody was used as a secondary antibody for the H9 IgG Ab. The
calculated number of HLA-A2/pp65 complexes inside cells (FIG. 10a) and on
the surface (FIG. 10b) as well as the number of general HLA-A2 complexes
inside the cells (FIG. 10c) and on the surface (FIG. 10d) in each time
scale, is shown for cells infected with AD169 (WT), RV798 (mutant), and
uninfected cells.

[0047] FIGS. 13a-j are confocal microscopy images of immuno-fluorescence
analyses depicting direct visualization of HLA-A2/pp65 complexes of the
surface (extracellular) of CMV infected fibroblasts. The cells were
extracellularly stained with the H9 Ab, and anti human alexa
fluor488 as a secondary Ab (FIGS. 13a-e). Noninfected fibroblast
cells were used as a control (FIGS. 13f-h). Verification of the virus
infection was with anti pp65 Ab and anti mouse alexa fluor594 as a
secondary Ab (FIGS. 13i-j).

[0048] FIGS. 14a-d depict the amino acid sequences (FIGS. 14a and c; SEQ
ID NOs:16 and 18) and the nucleic acid sequences (FIGS. 14b and d; SEQ ID
NOs:17 and 19) of the heavy chain (FIGS. 14a and b) and the light chain
(FIGS. 14c and d) of Fab H9. The CDRs are shown in red; the constant
regions are shown in green.

[0049] FIGS. 15a-d depict the amino acid sequences (FIGS. 15a and c; SEQ
ID NOs:20 and 22) and the nucleic acid sequences (FIGS. 15b and d; SEQ ID
NOs:21 and 23) of the heavy chain (FIGS. 15a and b) and the light chain
(FIGS. 15c and d) of Fab F5. The CDRs are shown in red; the constant
regions are shown in green.

[0050] FIGS. 16a-d are flow cytometry (FACS) analyses depicting the
detection of HLA-A2/pp65 complexes on the surface of virus-infected cells
taken from patients. PBMCs isolated from BMT recipients and healthy
donors were stained extracellular and intracellular with the H9 Ab and
the secondary anti human alexa fluor488 Ab. FIG. 16a-Confirmation of
the cells' typing by staining with anti HLA-A2 (BB7.2) Ab. FIG.
16b-Extracellular staining of the BMT recipient cells with the H9 Ab. No
detection of HLA-A2/pp65 complexes is seen in the infected cells using
the H9 Ab. FIGS. 16c-d-Intracellular staining of both BMT recipients
(FIG. 16c) and health donor cells (FIG. 16d) with the H9 Ab. A
significant specific staining with the H9 Ab of the permeabilized
infected cells is seen in the BMT recipients (FIG. 16c). In contrast, no
staining of the H9 Ab is seen in cells of the healthy control.

[0052] The present invention, in some embodiments thereof, relates to
antibodies capable of binding MHC molecules being complexed with
cytomegalovirus (CMV) pp65 or pp64 peptides which can be used to detect
CMV infection and presentation on the cell surface and, more
particularly, but not exclusively, to methods of diagnosing and treating
diseases associated with CMV infection.

[0053] Before explaining at least one embodiment of the invention in
detail, it is to be understood that the invention is not limited in its
application to the details set forth in the following description or
exemplified by the Examples. The invention is capable of other
embodiments or of being practiced or carried out in various ways. Also,
it is to be understood that the phraseology and terminology employed
herein is for the purpose of description and should not be regarded as
limiting.

[0054] While reducing the invention to practice, the present inventors
have generated human T cell receptor (TCR)-like antibodies directed
against complexes of MHC and CMV pp65 or pp64 antigenic peptides which
can recognize cells infected with CMV and thus can be used to diagnose
and treat diseases associated with CMV infection.

[0055] As shown in the Examples section which follows, recombinant
antibodies [e.g., clones H9 (the amino acid sequence of the heavy chain
is set forth by SEQ ID NO:16; the amino acid sequence of the light chain
is set forth by SEQ ID NO:18) and F5 (the amino acid sequence of the
heavy chain is set forth by SEQ ID NO:20; the amino acid sequence of the
light chain is set forth by SEQ ID NO:22] which can specifically
recognize MHC molecules when complexed with CMV pp65-derived peptides
such as the pp65495-503 (SEQ ID NO:3) were isolated and were found
to exhibit fine specificity to soluble or membrane-presented CMV pp65-MHC
class I complex (Examples 1 and 2 of the Examples section which follows).
In addition, multivalent forms of these antibodies (e.g., tetrameric Fabs
or bivalent IgG) which exhibit increased avidity while preserving the
specificity to the CMV pp65-MHC complex (Example 3 of the Examples
section which follows) were capable of detecting as low as 5% of
subpopulations of cells bearing CMV pp65 peptide-MHC complexes (Example 4
of the Examples section which follows). Cytotoxicity assays using
pp65-specific CD8+ T lymphocytes further demonstrated the specificity of
the TCR-like antibodies of the invention for CMV pp65-MHC complexes by
their ability to block killing by the CTLs (Example 6 of the Examples
section which follows). Moreover, the TCR-like antibodies of the
invention enabled one, for the first time, to follow CMV pp65-MHC class I
complexes both inside and on the surface of cells infected with CMV
(Example 5 of the Examples section which follows). In addition, as shown
in FIGS. 7-9 and described in Example 7 of the Examples section which
follows, the TCR-like antibodies of the invention demonstrated that there
is no correlation between class I MHC down regulation induced by
wild-type virus and the generation/presentation of the virus-specific
HLA-A2/pp65495-503 complex. Further quantitative data revealed that
specific HLA-A2/pp65 complexes are being generated in large amounts and
accumulated inside the infected cell in a mechanism that is independent
to the overall down regulation of HLA-A2 molecules in these cells
(Example 8 of the Examples section which follows). In addition, confocal
microscopy analysis demonstrated that immediately after CMV infection
specific HLA-A2/pp65 complexes are being generated and accumulated in the
Golgi compartment and only about 72 hours after infection are the
HLA-A2/pp65 complexes displayed on the cell surface (Example 9 of the
Examples section which follows). Moreover, as shown in FIGS. 16a-d and
described in Example 12 of the Examples section which follows, the
antibodies of the invention were shown to be capable of detecting
HLA-A2/pp65 complexes in blood cells of subjects with CMV reactivation
due to immune suppression (e.g., bone marrow transplanted subjects). In
addition, as shown in FIGS. 17a-j and described in Example 13 of the
Examples section which follows, incubation of cells with a proteasome
inhibitor resulted in increased presentation of the MHC/pp65 complexes on
the cell surface.

[0056] Thus, according to an aspect of some embodiments of the present
invention there is provided an antibody comprising an antigen recognition
domain capable of binding a Major histocompatibility complex (MHC)
molecule being complexed with a cytomegalovirus (CMV) pp65 or pp64
peptide, wherein the antibody does not bind the MHC molecule in an
absence of the complexed peptide, and wherein the antibody does not bind
the peptide in an absence of the MHC molecule.

[0057] As used herein, the phrase "major histocompatibility complex (MHC)"
refers to a complex of antigens encoded by a group of linked loci, which
are collectively termed H-2 in the mouse and HLA in humans. The two
principal classes of the MHC antigens, class I and class II, each
comprise a set of cell surface glycoproteins which play a role in
determining tissue type and transplant compatibility. In transplantation
reactions, cytotoxic T-cells (CTLs) respond mainly against foreign class
I glycoproteins, while helper T-cells respond mainly against foreign
class II glycoproteins.

[0058] Major histocompatibility complex (MHC) class I molecules are
expressed on the surface of nearly all cells. These molecules function in
presenting peptides which are mainly derived from endogenously
synthesized proteins to CD8+ T cells via an interaction with the
αβ T-cell receptor. The class I MHC molecule is a heterodimer
composed of a 46-kDa heavy chain which is non-covalently associated with
the 12-kDa light chain β-2 microglobulin. In humans, there are
several MHC haplotypes, such as, for example, HLA-A2, HLA-A1, HLA-A3,
HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45 and HLA-Cw8,
their sequences can be found at the kabbat data base, at
htexttransferprotocol://immuno.bme.nwu.edu. Further information
concerning MHC haplotypes can be found in Paul, B. Fundamental Immunology
Lippincott-Rven Press.

[0060] As used herein the term "peptide" refers to native peptides (either
proteolysis products or synthetically synthesized peptides) and further
to peptidomimetics, such as peptoids and semipeptoids which are peptide
analogs, which may have, for example, modifications rendering the
peptides more stable while in a body, or more immunogenic. Such
modifications include, but are not limited to, cyclization, N terminus
modification, C terminus modification, peptide bond modification,
including, but not limited to, CH2--NH, CH2--S,
CH2--S═O, O═C--NH, CH2--O, CH2--CH2,
S═C--NH, CH═CH or CF═CH, backbone modification and residue
modification. Methods for preparing peptidomimetic compounds are well
known in the art and are specified in Quantitative Drug Design, C. A.
Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is
incorporated by reference as if fully set forth herein. Further details
in this respect are provided hereinunder.

[0061] As used herein in the specification and in the claims section below
the term "amino acid" is understood to include the 20 naturally occurring
amino acids; those amino acids often modified post-translationally in
vivo, including for example hydroxyproline, phosphoserine and
phosphothreonine; and other unusual amino acids including, but not
limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,
nor-leucine and ornithine. Furthermore, the term "amino acid" includes
both D- and L-amino acids. Further elaboration of the possible amino
acids usable according to the invention and examples of non-natural amino
acids useful in MHC-1HLA-A2 recognizable peptide antigens are given
herein under.

[0062] Based on accumulated experimental data, it is nowadays possible to
predict which of the peptides of a protein will bind to MHC, class I. The
HLA-A2 MHC class I has been so far characterized better than other HLA
haplotypes, yet predictive and/or sporadic data is available for all
other haplotypes.

[0063] With respect to HLA-A2 binding peptides, assume the following
positions (P1-P9) in a 9-mer peptide:

P1-P2-P3-P4-P5-P6-P7-P8-P9

[0064] The P2 and P2 positions include the anchor residues which are the
main residues participating in binding to MHC molecules. Amino acid
resides engaging positions P2 and P9 are hydrophilic aliphatic
non-charged natural amino (examples being Ala, Val, Leu, Ile, Gln, Thr,
Ser, Cys, preferably Val and Leu) or of a non-natural hydrophilic
aliphatic non-charged amino acid [examples being norleucine (Nle),
norvaline (Nva), α-aminobutyric acid]. Positions P1 and P3 are also
known to include amino acid residues which participate or assist in
binding to MHC molecules, however, these positions can include any amino
acids, natural or non-natural. The other positions are engaged by amino
acid residues which typically do not participate in binding, rather these
amino acids are presented to the immune cells. Further details relating
to the binding of peptides to MHC molecules can be found in Parker, K.
C., Bednarek, M. A., Coligan, J. E., Scheme for ranking potential HLA-A2
binding peptides based on independent binding of individual peptide
side-chains. J Immunol. 152, 163-175, 1994, see Table V, in particular.
Hence, scoring of HLA-A2.1 binding peptides can be performed using the
HLA Peptide Binding Predictions software approachable through a worldwide
web interface at hypertexttransferprotocol://worldwideweb (dot) bimas
(dot) dcrt (dot) nih (dot) gov/molbio/hla_bind/index. This software is
based on accumulated data and scores every possible peptide in an
analyzed protein for possible binding to MHC HLA-A2.1 according to the
contribution of every amino acid in the peptide. Theoretical binding
scores represent calculated half-life of the HLA-A2.1-peptide complex.

[0065] Hydrophilic aliphatic natural amino acids at P2 and P9 can be
substituted by synthetic amino acids, preferably Nleu, Nval and/or
α-aminobutyric acid. P9 can be also substituted by aliphatic amino
acids of the general formula --HN(CH2)nCOOH, wherein n=3-5, as
well as by branched derivatives thereof, such as, but not limited to,

##STR00001##

wherein R is, for example, methyl, ethyl or propyl, located at any one or
more of the n carbons.

[0066] The amino terminal residue (position P1) can be substituted by
positively charged aliphatic carboxylic acids, such as, but not limited
to, H2N(CH2)nCOOH, wherein n=2-4 and
H2N--C(NH)--NH(CH2)nCOOH, wherein n=2-3, as well as by
hydroxy Lysine, N-methyl Lysine or ornithine (Orn). Additionally, the
amino terminal residue can be substituted by enlarged aromatic residues,
such as, but not limited to, H2N--(C6H6)--CH2--COOH,
p-aminophenyl alanine,
H2N--F(NH)--NH--(C6H6)--CH2--COOH, p-guanidinophenyl
alanine or pyridinoalanine (Pal). These latter residues may form hydrogen
bonding with the OH.sup.- moieties of the CMV pp65 residues at the MHC-1
N-terminal binding pocket, as well as to create, at the same time
aromatic-aromatic interactions.

[0067] Derivatization of amino acid residues at positions P4-P8, should
these residues have a side-chain, such as, OH, SH or NH2, like Ser,
Tyr, Lys, Cys or Orn, can be by alkyl, aryl, alkanoyl or aroyl. In
addition, OH groups at these positions may also be derivatized by
phosphorylation and/or glycosylation. These derivatizations have been
shown in some cases to enhance the binding to the T cell receptor.

[0068] Longer derivatives in which the second anchor amino acid is at
position P10 may include at P9 most L amino acids. In some cases shorter
derivatives are also applicable, in which the C terminal acid serves as
the second anchor residue.

[0069] Cyclic amino acid derivatives can engage position P4-P8, preferably
positions P6 and P7. Cyclization can be obtained through amide bond
formation, e.g., by incorporating Glu, Asp, Lys, Orn, di-amino butyric
(Dab) acid, di-aminopropionic (Dap) acid at various positions in the
chain (--CO--NH or --NH--CO bonds). Backbone to backbone cyclization can
also be obtained through incorporation of modified amino acids of the
formulas H--N((CH2)n--COOH)--C(R)H--COOH or
H--N((CH2)n--COOH)--C(R)H--NH2, wherein n=1-4, and further
wherein R is any natural or non-natural side chain of an amino acid.

[0070] Cyclization via formation of S--S bonds through incorporation of
two Cys residues is also possible. Additional side-chain to side chain
cyclization can be obtained via formation of an interaction bond of the
formula --(--CH2--)n--S--CH2--C--, wherein n=1 or 2, which
is possible, for example, through incorporation of Cys or homoCys and
reaction of its free SH group with, e.g., bromoacetylated Lys, Orn, Dab
or Dap.

[0072] These modifications can occur at any of the bonds along the peptide
chain and even at several (2-3) at the same time. According to some
embodiments of the invention, but not in all cases necessary, these
modifications should exclude anchor amino acids.

[0074] Various pp65 or pp64 MHC restricted peptides can be used to form
the MHC-CMV pp65 peptide complex. See for example, the peptides described
in Examples 10 and 11 of the Examples section which follows (Tables
5-137).

[0075] According to some embodiments of the invention, the antibodies
recognize a complex formed between the MHC class I molecule (HLA-A2) and
the CMV pp65 peptide set forth by SEQ ID NO:3.

[0076] The term "antibody" as used herein includes intact molecules as
well as functional fragments thereof, such as Fab, F(ab')2, Fv and
scFv that are capable of specific binding to a human major
histocompatibility complex (MHC) class I-restricted CMV pp65 or pp64
epitope. These functional antibody fragments are defined as follows: (i)
Fab, the fragment which contains a monovalent antigen-binding fragment of
an antibody molecule, can be produced by digestion of whole antibody with
the enzyme papain to yield an intact light chain and a portion of one
heavy chain; (ii) Fab', the fragment of an antibody molecule that can be
obtained by treating whole antibody with pepsin, followed by reduction,
to yield an intact light chain and a portion of the heavy chain; two Fab'
fragments are obtained per antibody molecule; (iii) F(ab')2, the
fragment of the antibody that can be obtained by treating whole antibody
with the enzyme pepsin without subsequent reduction; F(ab')2 is a
dimer of two Fab' fragments held together by two disulfide bonds; (iv)
Fv, defined as a genetically engineered fragment containing the variable
region of the light chain and the variable region of the heavy chain
expressed as two chains; and (v) scFv or "single chain antibody" ("SCA"),
a genetically engineered molecule containing the variable region of the
light chain and the variable region of the heavy chain, linked by a
suitable polypeptide linker as a genetically fused single chain molecule.

[0077] Methods of making these fragments are known in the art (See for
example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, New York, 1988, incorporated herein by reference) and
are further described herein below.

[0078] An exemplary method for generating antibodies capable of
specifically binding a CMV pp65 peptide restricted to an MHC-I complex is
described in the Examples section herein below.

[0079] In addition, such antibodies may be generated by (i) immunizing a
genetically engineered non-human mammal having cells expressing the human
major histocompatibility complex (MHC) class I, with a soluble form of an
MHC class I molecule being complexed with the HLA-restricted epitope;
(ii) isolating mRNA molecules from antibody producing cells, such as
splenocytes, of the non-human mammal; (iii) producing a phage display
library displaying protein molecules encoded by the mRNA molecules; and
(iv) isolating at least one phage clone from the phage display library,
the at least one phage displaying the antibody specifically bindable
(with an affinity below 200 nanomolar, e.g., below 100 nanomolar, e.g.,
below 50 nanomolar, e.g., below 30 nanomolar, e.g., below 20 nanomolar,
e.g., below 10 nanomolar) to the human major histocompatibility complex
(MHC) class I being complexed with the HLA-restricted epitope. The
genetic material of the phage isolate is then used to prepare a single
chain antibody or other forms of antibodies as is further described
herein below. For example, the genetic material of the phage isolate can
be used to prepare a single chain antibody which is conjugated to an
identifiable or a therapeutic moiety. According to some embodiments of
the invention, the non-human mammal is devoid of self MHC class I
molecules. According to some embodiments of the invention, the soluble
form of the MHC class I molecule is a single chain MHC class I
polypeptide including a functional human β-2 microglobulin amino
acid sequence directly or indirectly covalently linked to a functional
human MHC class I heavy chain amino acid sequence.

[0080] Recombinant MHC class I and class II complexes which are soluble
and which can be produced in large quantities are described in, for
example, Denkberg, G. et al. 2002, and further in U.S. patent application
Ser. No. 09/534,966 and PCT/IL01/00260 (published as WO 01/72768), all of
which are incorporated herein by reference. Soluble MHC class I molecules
are available or can be produced for any of the MHC haplotypes, such as,
for example, HLA-A2, HLA-A1, HLA-A3, HLA-A24, HLA-A28, HLA-A31, HLA-A33,
HLA-A34, HLA-B7, HLA-B45 and HLA-Cw8, following, for example the
teachings of PCT/IL01/00260, as their sequences are known and can be
found at the kabbat data base hypertexttransferprotocol://immuno (dot)
bme (dot) nwu (dot) edu/, the contents of the site is incorporated herein
by reference. Such soluble MHC class I molecules can be loaded with
suitable HLA-restricted epitopes and used for vaccination of non-human
mammal having cells expressing the human major histocompatibility complex
(MHC) class I as is further detailed hereinbelow.

[0082] Of particular interest is the paper by Pascolo et al., published in
J. Exp. Med. 185: 2043-2051, 1997, which describe the preparation of mice
expressing the human HLA-A2.1, H-2 Db and HHD MHC class I molecules and
devoid of mice MHC class I altogether.

[0083] An exemplary antibody, referred to as the H9 antibody, capable of
binding to an MHC class I complexed with a CMV pp65 epitope comprises
complementarity determining region (CDR) amino acid sequences as set
forth in SEQ ID NOs:24-26 (for the heavy chain) and 30-32 (for the light
chain).

[0084] Another exemplary antibody, referred to as the F5 antibody, capable
of binding to an MHC class I complexed with a CMV pp65 epitope comprises
complementarity determining region (CDR) amino acid sequences as set
forth in SEQ ID NOs:36-38 (for the heavy chain) and 42-44 (for the light
chain).

[0085] The invention provides a nucleic acid construct comprising a
nucleic acid sequence encoding the CDR sequences of the heavy chain and
the light chain of the antibody of the invention. The nucleic acid
construct may further comprise a promoter for directing expression of the
nucleic acid sequence in a host cell.

[0086] According to some embodiments of the invention, the nucleic acid
construct comprising the nucleic acid sequences set forth by SEQ ID
NOs:27-29 (for the heavy chain CDRs) and SEQ ID NOs:33-35 (for the light
chain CDRs).

[0087] According to some embodiments of the invention, the nucleic acid
construct comprising the nucleic acid sequences set forth by SEQ ID
NOs:39-41 (for the heavy chain CDRs) and SEQ ID NOs:45-47 (for the light
chain CDRs).

[0088] According to some embodiments of the invention, the nucleic acid
construct comprising the nucleic acid sequence set forth by SEQ ID NO:17
(for the heavy chain) and SEQ ID NO:19 (for the light chain).

[0089] According to some embodiments of the invention, the nucleic acid
construct comprising the nucleic acid sequence set forth by SEQ ID NO:21
(for the heavy chain) and SEQ ID NO:23 (for the light chain).

[0090] As mentioned herein above, the antibodies of the invention may be
antibody fragments. Antibody fragments according to the invention can be
prepared by proteolytic hydrolysis of the antibody or by expression in E.
coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other
protein expression systems) of a DNA sequence encoding the fragment.

[0091] Antibody fragments can be obtained by pepsin or papain digestion of
whole antibodies by conventional methods. For example, antibody fragments
can be produced by enzymatic cleavage of antibodies with pepsin to
provide a 5S fragment denoted F(ab')2. This fragment can be further
cleaved using a thiol reducing agent, and optionally a blocking group for
the sulfhydryl groups resulting from cleavage of disulfide linkages, to
produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic
cleavage using pepsin produces two monovalent Fab' fragments and an Fc
fragment directly. These methods are described, for example, by
Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references
contained therein, which patents are hereby incorporated by reference in
their entirety. See also Porter, R. R., Biochem. J., 73: 119-126, 1959.
Other methods of cleaving antibodies, such as separation of heavy chains
to form monovalent light-heavy chain fragments, further cleavage of
fragments, or other enzymatic, chemical, or genetic techniques may also
be used, so long as the fragments bind to the antigen that is recognized
by the intact antibody.

[0092] Fv fragments comprise an association of VH and VL chains.
This association may be noncovalent, as described in Inbar et al., Proc.
Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively, the variable chains
can be linked by an intermolecular disulfide bond or cross-linked by
chemicals such as glutaraldehyde. According to some embodiments of the
invention, the Fv fragments comprise VH and VL chains connected
by a peptide linker. These single-chain antigen binding proteins (scFv)
are prepared by constructing a structural gene comprising DNA sequences
encoding the VH and VL domains connected by an oligonucleotide.
The structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The recombinant
host cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing scFvs are described,
for example, by Whitlow and Filpula, Methods, 2: 97-105, 1991; Bird et
al., Science 242:423-426, 1988; Pack et al., Bio/Technology 11:1271-77,
1993; and Ladner et al., U.S. Pat. No. 4,946,778, which is hereby
incorporated by reference in its entirety.

[0093] Another form of an antibody fragment is a peptide coding for a
single complementarity-determining region (CDR). CDR peptides ("minimal
recognition units") can be obtained by constructing genes encoding the
CDR of an antibody of interest. Such genes are prepared, for example, by
using the polymerase chain reaction to synthesize the variable region
from RNA of antibody-producing cells. See, for example, Larrick and Fry,
Methods, 2: 106-10, 1991.

[0094] According to some embodiments of the invention, the antibodies are
multivalent forms such as tetrameric Fabs or IgG1 antibodies. The
advantages of the multivalent forms of the antibody of the invention
include increased avidity, yet without compromising the antibody
specificity to its target (i.e., the MHC-CMV pp65 peptide complex).
Exemplary methods for generating tetrameric Fabs or IgG1 antibodies are
described in the general materials and experimental methods of the
Examples section herein below.

[0095] Humanized forms of non-human (e.g., murine) antibodies are chimeric
molecules of immunoglobulins, immunoglobulin chains or fragments thereof
(such as Fv, Fab, Fab', F(ab')2 or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived from
non-human immunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are replaced by
residues from a CDR of a non-human species (donor antibody) such as
mouse, rat or rabbit having the desired specificity, affinity and
capacity. In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Humanized antibodies may also comprise residues which are found neither
in the recipient antibody nor in the imported CDR or framework sequences.
In general, the humanized antibody will comprise substantially all of at
least one, and typically two, variable domains, in which all or
substantially all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are those
of a human immunoglobulin consensus sequence.

[0097] Methods for humanizing non-human antibodies are well known in the
art. Generally, a humanized antibody has one or more amino acid residues
introduced into it from a source which is non-human. These non-human
amino acid residues are often referred to as import residues, which are
typically taken from an import variable domain. Humanization can be
essentially performed following the method of Winter and co-workers
[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature
332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by
substituting rodent CDRs or CDR sequences for the corresponding sequences
of a human antibody. Accordingly, such humanized antibodies are chimeric
antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the corresponding
sequence from a non-human species. In practice, humanized antibodies are
typically human antibodies in which some CDR residues and possibly some
FR residues are substituted by residues from analogous sites in rodent
antibodies.

[0099] It will be appreciated that once the CDRs of an antibody are
identified, using conventional genetic engineering techniques,
expressible polynucleotides encoding any of the forms or fragments of
antibodies described herein can be devised and modified in one of many
ways in order to produce a spectrum of related-products as further
described herein below.

[0100] The antibody of the invention can be used in vitro, ex vivo and in
vivo in various therapeutic or diagnostic applications.

[0101] In case the antibody of the invention is to be used for
administration into an individual (e.g., human), the human or humanized
antibody or antibody fragment will generally tend to be better tolerated
immunologically than one of non human origin since non variable portions
of non human antibodies will tend to trigger xenogeneic immune responses
more potent than the allogeneic immune responses triggered by human
antibodies which will typically be allogeneic with the individual. It
will be preferable to minimize such immune responses since these will
tend to shorten the half-life, and hence the effectiveness, of the
antibody of the invention in the individual. Furthermore, such immune
responses may be pathogenic to the individual, for example by triggering
harmful inflammatory reactions.

[0102] Alternately, an antibody or antibody fragment of human origin, or a
humanized antibody, will also be advantageous for applications in which a
functional physiological effect, for example an immune response against a
target cell, activated by a constant region of the antibody or antibody
fragment in the individual is desired. For example, for applications
including targeted cell killing a specific immune response is
advantageous. Such applications particularly include those in which the
functional interaction between a functional portion of the antibody or
antibody fragment, such as an Fc region, with a molecule such as an Fc
receptor or an Fc-binding complement component, is optimal when such a
functional portion is, similarly to the Fc region, of human origin.

[0103] Depending on the application and purpose, the antibody of the
invention which includes a constant region, or a portion thereof, of any
of various isotypes may be employed. According to some embodiments of the
invention, the isotype is selected so as to enable or inhibit a desired
physiological effect, or to inhibit an undesired specific binding of the
antibody of the invention via the constant region or portion thereof. For
example, for inducing antibody-dependent cell mediated cytotoxicity
(ADCC) by a natural killer (NK) cell, the isotype can be IgG; for
inducing ADCC by a mast cell/basophil, the isotype can be IgE; and for
inducing ADCC by an eosinophil, the isotype can be IgE or IgA. For
inducing a complement cascade the composition-of-matter may comprise an
antibody or antibody fragment comprising a constant region or portion
thereof capable of initiating the cascade. For example, the antibody or
antibody fragment may advantageously comprise a Cgamma2 domain of IgG or
Cmu3 domain of IgM to trigger a C1q-mediated complement cascade.

[0104] Conversely, for avoiding an immune response, such as the
aforementioned one, or for avoiding a specific binding via the constant
region or portion thereof, the antibody of the invention may not comprise
a constant region (be devoid of a constant region), or a portion thereof,
of the relevant isotype.

[0105] Additionally or alternatively, depending on the application and
purpose, the antibody or antibody fragment may be attached to any of
various functional moieties. An antibody or antibody fragment, such as
that of the invention, attached to a functional moiety may be referred to
in the art as an "immunoconjugate".

[0106] According to some embodiments of the invention, the functional
moiety is a detectable moiety or a toxin. An antibody or antibody
fragment attached to a toxin may be referred to in the art as an
"immunotoxin".

[0107] As is described and demonstrated in further detail hereinbelow, a
detectable moiety or a toxin may be particularly advantageously employed
in applications of the invention involving use of the antibody of the
invention to detect the complex or cells expressing the complex of the
MHC molecule and the cytomegalovirus (CMV) pp65 peptide and/or to kill
cells expressing or presenting such a complex.

[0108] For applications involving using the antibody of the invention to
detect the antigen-presenting portion of the complex, the detectable
moiety attached to the antibody or antibody fragment can be a reporter
moiety enabling specific detection of the MHC-CMV pp65 peptide complex
bound by the antibody or antibody fragment of the invention.

[0109] While various types of reporter moieties may be utilized to detect
the MHC-CMV pp65 peptide complex, depending on the application and
purpose, the reporter moiety can be a fluorophore or an enzyme.
Alternately, the reporter moiety may be a radioisotope, such as
[125]iodine. Further examples of reporter moieties, including those
detectable by Positron Emission Tomagraphy (PET) and Magnetic Resonance
Imaging (MRI), are well known to those of skill in the art.

[0114] As is described and illustrated in the Examples section below, the
antibody of the invention attached to a fluorophore, such as
phycoerythrin, can be used to optimally detect the MHC-CMV pp65 peptide
complex using various immunofluorescence-based detection methods.

[0115] Ample guidance regarding fluorophore selection, methods of linking
fluorophores to various types of molecules, such as an antibody or
antibody fragment of the invention, and methods of using such conjugates
to detect molecules which are capable of being specifically bound by
antibodies or antibody fragments comprised in such immunoconjugates is
available in the literature of the art [for example, refer to: Richard P.
Haugland, "Molecular Probes: Handbook of Fluorescent Probes and Research
Chemicals 1992-1994", 5th ed., Molecular Probes, Inc. (1994); U.S. Pat.
No. 6,037,137 to Oncoimmunin Inc.; Hermanson, "Bioconjugate Techniques",
Academic Press New York, N.Y. (1995); Kay M. et al., 1995. Biochemistry
34:293; Stubbs et al., 1996. Biochemistry 35:937; Gakamsky D. et al.,
"Evaluating Receptor Stoichiometry by Fluorescence Resonance Energy
Transfer," in "Receptors: A Practical Approach," 2nd ed., Stanford C. and
Horton R. (eds.), Oxford University Press, UK. (2001); U.S. Pat. No.
6,350,466 to Targesome, Inc.]. While various methodologies may be
employed to detect the MHC-CMV pp65 peptide complex using a fluorophore,
such detection is preferably effected as described and demonstrated in
the Examples section below.

[0116] Alternately, an enzyme may be advantageously utilized as the
detectable moiety to enable detection of the antigen-presenting portion
of the complex via any of various enzyme-based detection methods.
Examples of such methods include, but are not limited to, enzyme linked
immunosorbent assay (ELISA; for example, to detect the antigen-presenting
portion of the complex in a solution), enzyme-linked chemiluminescence
assay (for example, to detect the complex in an electrophoretically
separated protein mixture), and enzyme-linked immunohistochemical assay
(for example, to detect the complex in a fixed tissue).

[0117] Numerous types of enzymes may be employed to detect the
antigen-presenting portion of the complex, depending on the application
and purpose. For example, an antibody or antibody fragment attached to an
enzyme such as horseradish peroxidase can be used to effectively detect
the MHC-CMV pp65 peptide complex, such as via ELISA, or enzyme-linked
immunohistochemical assay.

[0120] The functional moiety may be attached to the antibody or antibody
fragment in various ways, depending on the context, application and
purpose.

[0121] A polypeptidic functional moiety, in particular a polypeptidic
toxin, may be advantageously attached to the antibody or antibody
fragment via standard recombinant techniques broadly practiced in the art
(for Example, refer to Sambrook et al., infra, and associated references,
listed in the Examples section which follows).

[0122] A functional moiety may also be attached to the antibody or
antibody fragment using standard chemical synthesis techniques widely
practiced in the art [for example, refer to the extensive guidelines
provided by The American Chemical Society (for example at:
hypertexttransferprotocol://worldwideweb (dot) chemistry (dot)
org/portal/Chemistry)]. One of ordinary skill in the art, such as a
chemist, will possess the required expertise for suitably practicing such
chemical synthesis techniques.

[0123] Alternatively, a functional moiety may be attached to the antibody
or antibody fragment by attaching an affinity tag-coupled antibody or
antibody fragment of the invention to the functional moiety conjugated to
a specific ligand of the affinity tag.

[0124] Various types of affinity tags may be employed to attach the
antibody or antibody fragment to the functional moiety.

[0125] Examples of detectable moieties that can be used in the invention
include but are not limited to radioactive isotopes, phosphorescent
chemicals, chemiluminescent chemicals, fluorescent chemicals, enzymes,
fluorescent polypeptides and epitope tags. The detectable moiety can be a
member of a binding pair, which is identifiable via its interaction with
an additional member of the binding pair, and a label which is directly
visualized. In one example, the member of the binding pair is an antigen
which is identified by a corresponding labeled antibody. In one example,
the label is a fluorescent protein or an enzyme producing a colorimetric
reaction.

[0126] When the detectable moiety is a polypeptide, the immunolabel (i.e.
the antibody conjugated to the detectable moiety) may be produced by
recombinant means or may be chemically synthesized by, for example, the
stepwise addition of one or more amino acid residues in defined order
using solid phase peptide synthetic techniques. Examples of polypeptide
detectable moieties that can be linked to the antibodies of the invention
using recombinant DNA technology include fluorescent polypeptides,
phosphorescent polypeptides, enzymes and epitope tags.

[0127] Expression vectors can be designed to fuse proteins encoded by the
heterologous nucleic acid insert to fluorescent polypeptides. For
example, antibodies can be expressed from an expression vector fused with
a green fluorescent protein (GFP)-like polypeptide. A wide variety of
vectors are commercially available that fuse proteins encoded by
heterologous nucleic acids to the green fluorescent protein from Aequorea
victoria ("GFP"), the yellow fluorescent protein and the red fluorescent
protein and their variants (e.g., Evrogen). In these systems, the
fluorescent polypeptide is entirely encoded by its amino acid sequence
and can fluoresce without requirement for cofactor or substrate.
Expression vectors that can be employed to fuse proteins encoded by the
heterologous nucleic acid insert to epitope tags are commercially
available (e.g., BD Biosciences, Clontech).

[0128] Alternatively, chemical attachment of a detectable moiety to the
antibodies of the invention can be effected using any suitable chemical
linkage, direct or indirect, as via a peptide bond (when the detectable
moiety is a polypeptide), or via covalent bonding to an intervening
linker element, such as a linker peptide or other chemical moiety, such
as an organic polymer. Such chimeric peptides may be linked via bonding
at the carboxy (C) or amino (N) termini of the peptides, or via bonding
to internal chemical groups such as straight, branched or cyclic side
chains, internal carbon or nitrogen atoms, and the like. Such modified
peptides can be easily identified and prepared by one of ordinary skill
in the art, using well known methods of peptide synthesis and/or covalent
linkage of peptides. Description of fluorescent labeling of antibodies is
provided in details in U.S. Pat. Nos. 3,940,475, 4,289,747, and
4,376,110.

[0129] Exemplary methods for conjugating two peptide moieties are
described herein below:

[0130] SPDP Conjugation:

[0131] Any SPDP conjugation method known to those skilled in the art can
be used. For example, in one illustrative embodiment, a modification of
the method of Cumber et al. (1985, Methods of Enzymology 112: 207-224) as
described below, is used.

[0132] A peptide, such as an identifiable or therapeutic moiety, (1.7
mg/ml) is mixed with a 10-fold excess of SPDP (50 mM in ethanol) and the
antibody is mixed with a 25-fold excess of SPDP in 20 mM sodium
phosphate, 0.10 M NaCl pH 7.2 and each of the reactions incubated, e.g.,
for 3 hours at room temperature. The reactions are then dialyzed against
PBS.

[0133] The peptide is reduced, e.g., with 50 mM DTT for 1 hour at room
temperature. The reduced peptide is desalted by equilibration on G-25
column (up to 5% sample/column volume) with 50 mM KH2PO4 pH
6.5. The reduced peptide is combined with the SPDP-antibody in a molar
ratio of 1:10 antibody:peptide and incubated at 4° C. overnight to
form a peptide-antibody conjugate.

[0134] Glutaraldehyde Conjugation:

[0135] Conjugation of a peptide (e.g., an identifiable or therapeutic
moiety) with an antibody can be accomplished by methods known to those
skilled in the art using glutaraldehyde. For example, in one illustrative
embodiment, the method of conjugation by G. T. Hermanson (1996, "Antibody
Modification and Conjugation, in Bioconjugate Techniques, Academic Press,
San Diego) described below, is used.

[0136] The antibody and the peptide (1.1 mg/ml) are mixed at a 10-fold
excess with 0.05% glutaraldehyde in 0.1 M phosphate, 0.15 M NaCl pH 6.8,
and allowed to react for 2 hours at room temperature. 0.01 M lysine can
be added to block excess sites. After-the reaction, the excess
glutaraldehyde is removed using a G-25 column equilibrated with PBS (10%
v/v sample/column volumes)

[0137] Carbodiimide Conjugation:

[0138] Conjugation of a peptide with an antibody can be accomplished by
methods known to those skilled in the art using a dehydrating agent such
as a carbodiimide. Most preferably the carbodiimide is used in the
presence of 4-dimethyl aminopyridine. As is well known to those skilled
in the art, carbodiimide conjugation can be used to form a covalent bond
between a carboxyl group of peptide and an hydroxyl group of an antibody
(resulting in the formation of an ester bond), or an amino group of an
antibody (resulting in the formation of an amide bond) or a sulfhydryl
group of an antibody (resulting in the formation of a thioester bond).

[0139] Likewise, carbodiimide coupling can be used to form analogous
covalent bonds between a carbon group of an antibody and an hydroxyl,
amino or sulfhydryl group of the peptide. See, generally, J. March,
Advanced Organic Chemistry: Reaction's, Mechanism, and Structure, pp.
349-50 & 372-74 (3d ed.), 1985. By means of illustration, and not
limitation, the peptide is conjugated to an antibody via a covalent bond
using a carbodiimide, such as dicyclohexylcarbodiimide. See generally,
the methods of conjugation by B. Neises et al. (1978, Angew Chem., Int.
Ed. Engl. 17:522; A. Hassner et al. (1978, Tetrahedron Lett. 4475); E. P.
Boden et al. (1986, J. Org. Chem. 50:2394) and L. J. Mathias (1979,
Synthesis 561). The level of immunocomplex may be compared to a control
sample from a non-diseased subject, wherein an up-regulation of
immunocomplex formation is indicative of disease associated with CMV
infection. Preferably, the subject is of the same species e.g. human,
preferably matched with the same age, weight, sex etc. It will be
appreciated that the control sample may also be of the same subject from
a healthy tissue, prior to disease progression or following disease
remission.

[0140] Preferably, the affinity tag is a biotin molecule, more preferably
a streptavidin molecule.

[0141] A biotin or streptavidin affinity tag can be used to optimally
enable attachment of a streptavidin-conjugated or a biotin-conjugated
functional moiety, respectively, to the antibody or antibody fragment due
to the capability of streptavidin and biotin to bind to each other with
the highest non covalent binding affinity (i.e., with a Kd of about
10-14 to 10-15). A biotin affinity tag may be highly
advantageous for applications benefiting from. Thus, the antibody of
invention can be a multimeric form of the antibody or antibody fragment,
which may be optimally formed by conjugating multiple biotin-attached
antibodies or antibody fragments of the invention to a streptavidin
molecule, as described in further detail below.

[0142] As used herein the term "about" refers to plus or minus 10 percent.

[0143] Various methods, widely practiced in the art, may be employed to
attach a streptavidin or biotin molecule to a molecule such as the
antibody or antibody fragment to a functional moiety.

[0144] For example, a biotin molecule may be advantageously attached to an
antibody or antibody fragment of the invention attached to a recognition
sequence of a biotin protein ligase. Such a recognition sequence is a
specific polypeptide sequence serving as a specific biotinylation
substrate for the biotin protein ligase enzyme. Ample guidance for
biotinylating a target polypeptide such as an antibody fragment using a
recognition sequence of a biotin protein ligase, such as the recognition
sequence of the biotin protein ligase BirA, is provided in the literature
of the art (for example, refer to: Denkberg, G. et al., 2000. Eur. J.
Immunol. 30:3522-3532). Preferably, such biotinylation of the antibody or
antibody fragment is effected as described and illustrated in the
Examples section below.

[0147] As mentioned, the antibody may be conjugated to a therapeutic
moiety. The therapeutic moiety can be, for example, a cytotoxic moiety, a
toxic moiety, a cytokine moiety and a second antibody moiety comprising a
different specificity to the antibodies of the invention.

[0148] In a similar fashion to an immunolabel, an immunotoxin (i.e. a
therapeutic moiety attached to an antibody of the invention) may be
generated by recombinant or non-recombinant means. Thus, the invention
envisages a first and second polynucleotide encoding the antibody of the
invention and the therapeutic moiety, respectively, ligated in frame, so
as to encode an immunotoxin. The following Table 1 provides examples of
sequences of therapeutic moieties.

[0149] According to some embodiments of the invention, the toxic moiety is
PE38 KDEL.

[0150] Exemplary methods of conjugating the antibodies of the invention to
peptide therapeutic agents are described herein above.

[0151] As mentioned, the antibody of the invention, which is capable of
specifically recognizing and binding an MHC-CMV pp65 peptide complex as
described above, can be used to detecting cell expressing a
cytomegalovirus (CMV) antigen.

[0152] Thus, according to an aspect of some embodiments of the invention
there is provided a method of detecting a cell expressing a
cytomegalovirus (CMV) antigen. The method is effected by contacting the
cell with the antibody under conditions which allow immunocomplex
formation, wherein a presence or a level above a predetermined threshold
of the immunocomplex is indicative of CMV expression in the cell.

[0153] The contacting may be effected in vitro (e.g., in a cell line), ex
vivo or in vivo.

[0154] As mentioned, the method of the invention is effected under
conditions sufficient to form an immunocomplex (e.g. a complex between
the antibodies of the invention and the MHC-CMV pp65 peptide); such
conditions (e.g., appropriate concentrations, buffers, temperatures,
reaction times) as well as methods to optimize such conditions are known
to those skilled in the art, and examples are disclosed herein.

[0155] As described in the Examples section which follows, the
immunocomplex can be formed and detected within the cell or on the cell
surface. For detection in the cell, the conditions include a
permeabilization agent (e.g., a solution including saponin), to enable
penetration of the antibody inside the cell. According to some
embodiments of the invention, the immunocomplex is formed on the surface
of the cell.

[0156] Determining a presence or level of the immunocomplex of the
invention is dependent on the detectable moiety to which the antibody is
attached, essentially as described hereinabove.

[0157] A non-limiting example of the immunocomplex of the invention is the
complex formed between the antibody of the invention (e.g., H9 or F5) and
a protein complex comprising MHC class I heavy chain (HLA-A2) and pp65
peptide as set forth by SEQ ID NO:3.

[0158] As mentioned, the antibody of the invention, which is capable of
specifically recognizing and binding an MHC-CMV pp65 peptide complex, can
be used to diagnose CMV infection in a subject in need thereof.

[0159] Thus, according to another aspect of the invention, there is
provided a method of diagnosing a cytomegalovirus (CMV) infection in a
subject in need thereof. The method is effected by contacting a cell of
the subject with the antibody under conditions which allow immunocomplex
formation, wherein a presence or a level above a pre-determined threshold
of the immunocomplex in the cell is indicative of the CMV infection in
the subject.

[0160] As used herein the phrase "subject in need thereof" refers to a
mammal, preferably, a human subject which is suspected of being infected
with CMV.

[0161] According to some embodiments of the invention, the subject has a
suppressed or a compromised immune system, such as an immuno-compromised
organ transplant recipient or a subject infected with human
immunodeficiency virus (HIV).

[0162] According to some embodiments of the invention, the CMV infection
is associated with a disease selected from the group consisting of
mononucleosis, retinitis, pneumonia, gastrointestinal disorders, and
encephalitis.

[0163] According to some embodiments of the invention, the cell is a
retina cell, lung epithelial cell, a gastrointestinal epithelial cell
and/or a brain cell.

[0164] The antibody described herein can be used to treat a disease
associated with CMV infection.

[0165] According to an additional aspect of the invention there is
provided a method of treating a disease associated with cytomegalovirus
(CMV) infection, the method is effected by administering to a subject in
need thereof a therapeutically effective amount of the antibody thereby
treating the disease associated with CMV infection.

[0166] The term "treating" refers to inhibiting or arresting the
development of a disease, disorder or condition and/or causing the
reduction, remission, or regression of a disease, disorder or condition.
Those of skill in the art will understand that various methodologies and
assays can be used to assess the development of a disease, disorder or
condition, and similarly, various methodologies and assays may be used to
assess the reduction, remission or regression of a disease, disorder or
condition.

[0167] The antibodies of the invention may be provided per se or may be
administered as a pharmaceutical composition.

[0168] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described herein
with other chemical components such as physiologically suitable carriers
and excipients. The purpose of a pharmaceutical composition is to
facilitate administration of a compound to an organism.

[0169] Herein the term "active ingredient" refers to the antibodies of the
invention accountable for the biological effect.

[0170] Hereinafter, the phrases "physiologically acceptable carrier" and
"pharmaceutically acceptable carrier" which may be interchangeably used
refer to a carrier or a diluent that does not cause significant
irritation to an organism and does not abrogate the biological activity
and properties of the administered compound. An adjuvant is included
under these phrases.

[0171] Herein the term "excipient" refers to an inert substance added to a
pharmaceutical composition to further facilitate administration of an
active ingredient. Examples, without limitation, of excipients include
calcium carbonate, calcium phosphate, various sugars and types of starch,
cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0172] Techniques for formulation and administration of drugs may be found
in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton,
Pa., latest edition, which is incorporated herein by reference.

[0174] Alternately, one may administer the pharmaceutical composition in a
local rather than systemic manner, for example, via injection of the
pharmaceutical composition directly into a tissue region of a patient.

[0175] Pharmaceutical compositions of the invention may be manufactured by
processes well known in the art, e.g., by means of conventional mixing,
dissolving, granulating, dragee-making, levigating, emulsifying,
encapsulating, entrapping or lyophilizing processes.

[0176] Pharmaceutical compositions for use in accordance with the
invention thus may be formulated in conventional manner using one or more
physiologically acceptable carriers comprising excipients and
auxiliaries, which facilitate processing of the active ingredients into
preparations which, can be used pharmaceutically. Proper formulation is
dependent upon the route of administration chosen.

[0177] For injection, the active ingredients of the pharmaceutical
composition may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution, Ringer's
solution, or physiological salt buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.

[0178] For oral administration, the pharmaceutical composition can be
formulated readily by combining the active compounds with
pharmaceutically acceptable carriers well known in the art. Such carriers
enable the pharmaceutical composition to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the
like, for oral ingestion by a patient. Pharmacological preparations for
oral use can be made using a solid excipient, optionally grinding the
resulting mixture, and processing the mixture of granules, after adding
suitable auxiliaries if desired, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,
for example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose,
sodium carbomethylcellulose; and/or physiologically acceptable polymers
such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may
be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic
acid or a salt thereof such as sodium alginate.

[0179] Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar solutions may be used which may optionally contain gum
arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol,
titanium dioxide, lacquer solutions and suitable organic solvents or
solvent mixtures. Dyestuffs or pigments may be added to the tablets or
dragee coatings for identification or to characterize different
combinations of active compound doses.

[0180] Pharmaceutical compositions which can be used orally, include
push-fit capsules made of gelatin as well as soft, sealed capsules made
of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit
capsules may contain the active ingredients in admixture with filler such
as lactose, binders such as starches, lubricants such as talc or
magnesium stearate and, optionally, stabilizers. In soft capsules, the
active ingredients may be dissolved or suspended in suitable liquids,
such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In
addition, stabilizers may be added. All formulations for oral
administration should be in dosages suitable for the chosen route of
administration.

[0181] For buccal administration, the compositions may take the form of
tablets or lozenges formulated in conventional manner.

[0182] For administration by nasal inhalation, the active ingredients for
use according to the invention are conveniently delivered in the form of
an aerosol spray presentation from a pressurized pack or a nebulizer with
the use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In
the case of a pressurized aerosol, the dosage unit may be determined by
providing a valve to deliver a metered amount. Capsules and cartridges
of, e.g., gelatin for use in a dispenser may be formulated containing a
powder mix of the compound and a suitable powder base such as lactose or
starch.

[0183] The pharmaceutical composition described herein may be formulated
for parenteral administration, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit dosage
form, e.g., in ampoules or in multidose containers with optionally, an
added preservative. The compositions may be suspensions, solutions or
emulsions in oily or aqueous vehicles, and may contain formulatory agents
such as suspending, stabilizing and/or dispersing agents.

[0184] Pharmaceutical compositions for parenteral administration include
aqueous solutions of the active preparation in water-soluble form.
Additionally, suspensions of the active ingredients may be prepared as
appropriate oily or water based injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame oil, or
synthetic fatty acids esters such as ethyl oleate, triglycerides or
liposomes. Aqueous injection suspensions may contain substances, which
increase the viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension may also
contain suitable stabilizers or agents which increase the solubility of
the active ingredients to allow for the preparation of highly
concentrated solutions.

[0185] Alternatively, the active ingredient may be in powder form for
constitution with a suitable vehicle, e.g., sterile, pyrogen-free water
based solution, before use.

[0186] The pharmaceutical composition of the invention may also be
formulated in rectal compositions such as suppositories or retention
enemas, using, e.g., conventional suppository bases such as cocoa butter
or other glycerides.

[0187] Pharmaceutical compositions suitable for use in context of the
invention include compositions wherein the active ingredients are
contained in an amount effective to achieve the intended purpose. More
specifically, a therapeutically effective amount means an amount of
active ingredients (the antibody of the invention or the nucleic acid
construct encoding same) effective to prevent, alleviate or ameliorate
symptoms of a pathology, (e.g., a disease associated with cytomegalovirus
infection) or prolong the survival of the subject being treated.

[0188] Determination of a therapeutically effective amount is well within
the capability of those skilled in the art, especially in light of the
detailed disclosure provided herein.

[0189] For any preparation used in the methods of the invention, the
therapeutically effective amount or dose can be estimated initially from
in vitro and cell culture assays. For example, a dose can be formulated
in animal models to achieve a desired concentration or titer. Such
information can be used to more accurately determine useful doses in
humans.

[0190] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical procedures
in vitro, in cell cultures or experimental animals. The data obtained
from these in vitro and cell culture assays and animal studies can be
used in formulating a range of dosage for use in human. The dosage may
vary depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of administration
and dosage can be chosen by the individual physician in view of the
patient's condition. (See e.g., Fingl, et al., 1975, in "The
Pharmacological Basis of Therapeutics", Ch. 1 p. 1).

[0191] Dosage amount and interval may be adjusted individually to provide
plasma or brain levels of the active ingredient are sufficient to induce
or suppress the biological effect (minimal effective concentration, MEC).
The MEC will vary for each preparation, but can be estimated from in
vitro data. Dosages necessary to achieve the MEC will depend on
individual characteristics and route of administration. Detection assays
can be used to determine plasma concentrations.

[0192] Depending on the severity and responsiveness of the condition to be
treated, dosing can be of a single or a plurality of administrations,
with course of treatment lasting from several days to several weeks or
until cure is effected or diminution of the disease state is achieved.

[0193] The amount of a composition to be administered will, of course, be
dependent on the subject being treated, the severity of the affliction,
the manner of administration, the judgment of the prescribing physician,
etc.

[0194] Compositions of the invention may, if desired, be presented in a
pack or dispenser device, such as an FDA approved kit, which may contain
one or more unit dosage forms containing the active ingredient. The pack
may, for example, comprise metal or plastic foil, such as a blister pack.
The pack or dispenser device may be accompanied by instructions for
administration and use. The pack or dispenser may also be accommodated by
a notice associated with the container in a form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals, which notice is reflective of approval by the agency of
the form of the compositions or human or veterinary administration. Such
notice, for example, may be of labeling approved by the U.S. Food and
Drug Administration for prescription drugs or of an approved product
insert. Compositions comprising a preparation of the invention formulated
in a compatible pharmaceutical carrier may also be prepared, placed in an
appropriate container, and labeled for treatment of an indicated
condition, as if further detailed above.

[0195] As used herein the term "about" refers to ±10%.

[0196] It is appreciated that certain features of the invention, which
are, for clarity, described in the context of separate embodiments, may
also be provided in combination in a single embodiment. Conversely,
various features of the invention, which are, for brevity, described in
the context of a single embodiment, may also be provided separately or in
any suitable subcombination or as suitable in any other described
embodiment of the invention. Certain features described in the context of
various embodiments are not to be considered essential features of those
embodiments, unless the embodiment is inoperative without those elements.

[0197] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below find
experimental support in the following examples.

EXAMPLES

[0198] Reference is now made to the following examples, which together
with the above descriptions, illustrate the invention in a non limiting
fashion.

[0200] Single-chain MHC (scMHC)/peptide complexes were produced by in
vitro refolding of inclusion bodies produced in Escherichia coli, as
described (Denkberg, G. et al., 2000). Briefly, a single-chain
β2-microglobulin (β2m)-HLA/A2 (scMHC) construct, in
which the β2m and HLA-A2 genes are connected to each other by a
flexible peptide linker (wherein the β2m gene is translationally
fused upstream of the gene encoding the MHC heavy chain) (HLA-A2), was
engineered to contain the BirA recognition sequence for site-specific
biotinylation at the C terminus (scMHC-BirA). In vitro refolding was
performed in the presence of a 5-10 molar excess of the antigenic
peptides, as described (Denkberg, G. et al., 2000). Correctly folded
MHC/peptide complexes were isolated and purified by anion exchange
Q-Sepharose chromatography (Pharmacia, Peapack, N.J.), followed by
site-specific biotinylation using the BirA enzyme (Avidity, Denver,
Colo.), as previously described (Altman, J. D. et al., 1996). The
homogeneity and purity of the scMHC-peptide complexes were analyzed by
various biochemical means, including SDS-PAGE, size exclusion
chromatography, and ELISA, as previously described (Denkberg, G. et al.,
2000).

[0201] Selection of Phage Antibodies on Biotinylated Complexes

[0202] Selection of phage Abs on biotinylated complexes was preformed, as
described (Denkberg, G., et al., 2002; Lev A., et al., 2002). Briefly, a
large human Fab library containing 3.7×1010 different Fab
clones (De Haard, H J., et al., 1999) was used for the selection. Phages
(1013) were first preincubated with streptavidin-coated paramagnetic
beads (200 μl; Dynal, Oslo, Norway) to deplete the streptavidin
binders. The remaining phages were subsequently used for panning with
decreasing amounts of biotinylated scMHC-peptide complexes. The
streptavidin-depleted library was incubated in solution with soluble
biotinylated scHLA-A2/pp65 complexes (500 nM for the first round, and 100
nM for the following rounds) for 30 minutes at room temperature (RT).

[0203] Streptavidin-coated magnetic beads (200 μl for the first round
of selection, and 100 μl for the second and third rounds) were added
to the mixture and incubated for 10-15 minutes at RT. The beads were
washed extensively 12 times with PBS/Tween 0.1%, and additional two
washes were with PBS. Bound phages were eluted with triethylamine (100
mM, 5 minutes at RT), followed by neutralization with Tris-HCl (1 M, pH
7.4), and used to infect E. coli TG1 cells (OD=0.5) for 30 minutes at
37° C.

[0204] The diversity of the selected Abs was determined by DNA
fingerprinting using a restriction endonuclease (BstNI), which is a
frequent cutter of Ab V gene sequences. The Fab DNA of different clones
was PCR amplified using the primers pUC-reverse
[5'-AGCGGATAACAATTTCACACAGG-3' (SEQ ID NO:1)] and fd-tet-seq24
[5'-TTTGTCGTCTTTCCAGACGTTAGT-3' (SEQ ID NO:2)], followed by digestion
with BstNI (NEB, Beverly, Mass.) (2 hours, 60° C.) and analysis on
agarose gel electrophoresis.

[0205] Expression and Purification of Soluble Recombinant Fab Abs

[0206] Fab Abs were expressed and purified, as described recently
(Denkberg, G., et al., 2000). BL21 bacterial cells were grown to
OD600=0.8-1.0 and induced to express the recombinant Fab Ab by the
addition of 1 mM isopropyl 13-D-thiogalactoside (IPTG) for 3-4 hours at
30° C. Periplasmic content was released using the B-PER solution
(Pierce, Rockford, Ill.), which was applied onto a prewashed TALON column
(Clontech, Palo Alto, Calif.). Bound Fabs were eluted using 0.5 ml of 100
mM imidazole in PBS. The eluted Fabs were dialyzed twice against PBS
(overnight, 4° C.) to remove residual imidazole.

[0207] ELISA with Phage Clones and Purified Fab Abs

[0208] The binding specificities of individual phage clones and soluble
Fab were determined by ELISA using biotinylated scMHC-peptide complexes.
ELISA plates (Falcon) were coated overnight with BSA-biotin (1
μg/well). After having been washed, the plates were incubated (1 hour,
RT) with streptavidin (1 μg/well), washed extensively, and further
incubated (1 hour, RT) with 0.5 μg of MHC/peptide complexes. The
plates were blocked for 30 minutes at RT with PBS/2% skim milk and
subsequently were incubated for 1 hour at RT with phage clones
(˜109 phages/well) or various concentrations of soluble
purified Fab. After having been washed, the plates were incubated with
HRP-conjugated/anti-human Fab Ab (for soluble Fabs) or HRP-conjugated
anti-M13 phage (for phage-displayed Fabs). Detection was performed using
tetramethylbenzidine reagent (Sigma-Aldrich, St. Louis, Mo.). The
HLA-A2-restricted peptides used for specificity studies of the Fab phage
clones or purified Fab Abs are as described in Examples 1 and 2 below.

[0209] Generation of Fluorescently-Labeled Tetrameric Fab

[0210] The genes encoding the L and H chain of Fab H9 were cloned
separately into a T7-promotor pET-based expression vector. The L chain
gene was engineered to contain the BirA recognition sequence for
site-specific biotinylation at the C terminus. These constructs were
expressed separately in E. coli BL21 cells and upon induction with IPTG,
intracellular inclusion bodies that contain large amounts of the
recombinant protein accumulated. Inclusion bodies of both chains were
purified, solubilized, reduced with 10 mg/ml DTE (Dithioerithrol), and
subsequently refolded at a 1:1 ratio in a redox-shuffling buffer system
containing 0.1 M Tris, 0.5 M arginine, and 0.09 mM oxidized glutathione,
pH 8.0. Correctly folded Fab was then isolated and purified by anion
exchange MonoQ chromatography (Pharmacia). The Fab peak fractions were
concentrated using Centricon-30 (Amicon, Beverly, Mass.) to 1 mg/ml, and
the buffer was exchanged to Tris-HCl (10 mM, pH 8.0). Biotinylation was
performed using the BirA enzyme (Avidity), as previously described.
Excess biotin was removed from biotinylated Fabs using a G-25 desalting
column. PE-labeled streptavidin (Jackson ImmunoResearch, West Grove, Pa.)
was added at a molar ratio of 1:4 to produce fluorescent tetramers of the
biotinylated Fab.

[0211] Generation of Whole IgG from Recombinant Fab

[0212] To transform the recombinant fragments into whole IgG molecules,
the eukaryotic expression vector pCMV/myc/ER (Invitrogen) was used. The
heavy and the light chains of the Fab were cloned separately. Each
shuttle expression vector carries a different antibiotics resistance gene
and thus expression was facilitated by co-transfection of the two
constructs into human embryonic kidney HEK293 cells. Cotransfections of
HEK293 cells were performed using the nonliposomal transfection reagent
FuGene 6 (Roche, Brussels, Belgium) according to the manufacturer's
instructions. The transfection was performed with serum free medium
containing 0.8 mg/ml of G418, and 100 μg/ml of hygromycin. Forty-eight
hours after transfection limiting dilutions were performed into medium
containing 0.8 mg/ml of G418, and 100 μg/ml of hygromycin. Cells were
plated in 96-well plates at 1000 cells per well. Medium was exchanged
after 5 and 10 days. Wells in which a single colony grew up to 50% of the
well were further trypsinized with 20 μl and 20 μl medium and
splitted into two wells: 10 μl into a 24 well plate (backup) and 30
μl into a 24 well plate (experiment). When the plate reached 80%
confluency, serum starvation was initiated by reducing each day serum
percentile to 0.5%. After 48 hours of incubation with 0.5% fetal calf
serum (FCS), screening of cell culture supernatants was performed by
ELISA and FACS assays. The IgG secreting clones that exhibited the best
binding reactivity as detected by ELISA, FACS and the highest amount of
protein, were selected for antibody production and purification. Protein
A-Sepharose® 4 Fast Flow beads (Amersham) were prepared according to
the manufacturer's instructions. Briefly, supernatant was loaded on the
Protein A-Sepharose beads at 15-50 ml/h. Unbound immunoglobulins were
washed with 0.001 M NaH2PO4 and 0.019 M Na2HPO4.
Bound immunoglobulins were then eluted with 0.1 M citric acid at pH 3.
Five fractions were collected with 250 μl of elusion buffer and
immediately neutralized with 80 μl of Tris-HCL pH 9. IgG concentration
was measured using the Pierce protein assay. The eluted protein was
dialyzed against PBS pH 7.4 over night. 10 mgs of IgG were produced from
1 L of culture supernatant.

[0213] Flow Cytometry

[0214] The B cell line RMAS-HHD, which is transfected with a single-chain
β2m-HLA-A2 gene, the EBV-transformed HLA-A2.sup.+ JY cells, and
the HLA-A2-B cell line APD-70 were used to determine the reactivity of
the recombinant Fab Abs with cell surface-expressed HLA-A2/peptide
complexes. Peptide pulsing was performed as indicated: 106 cells
were washed twice with serum-free RPMI and incubated overnight at
26° C. or 37° C., respectively, in medium containing 1-50
μM of the peptide. The RMAS-HHD cells were subsequently incubated at
37° C. for 2-3 hours to stabilize cell surface expression of
MHC-peptide complexes.

[0215] Cells were incubated for 60 minutes at 4° C. with
recombinant Fab Abs (10 μg/ml) in 100 μl PBS. After one wash, the
cells were incubated with 1 μg anti-human Fab (Jackson ImmunoResearch)
for another 60 minutes at 4° C. After three washes, the cells were
resuspended in ice-cold PBS. The cells were analyzed by a FACStar flow
cytometer (BD Biosciences, San Jose, Calif.).

[0216] Surface Plasmon Resonance

[0217] 0.0025 mg/ml of biotinylated HLA-A2/pp65 or control HLA-A2/EBV
complexes were bound to a streptavidin (SA) sensor chip (Biacore,
Uppsala, Sweden) per well. Measurements of 780-800 RU were detected for
each well after complexes binding. Soluble isolated antibodies in their
monomeric/IgG form were diluted in PBS at three concentration (0.05
μM, 0.1 μM, 0.2 μM) and were flowed over the relevant wells at a
rate of 10 μl/min at room temperature. Responses were recorded using
Biacore 2000 and analyzed using BIAevaluation software 3.2 (Biacore,
Uppsala, Sweden).

[0221] Target cells were cultured in 48-well plates in DMEM medium plus
10% FCS and were grown up until confluent. Cells were washed and
incubated overnight with 15 μCi/ml (1Ci 37 GBq) [35S] methionine
(NEN). After 1 hour of incubation with the IgG H9 (10-20 μg/ml or the
indicated concentration at 37° C.), effector CTL cells were added
at a target:effector ratio of 1:3 respectively and incubated for 5 hours
at 37° C. After incubation, [35S] methionine release from
target cells was measured in a 50-n1 sample of the culture supernatant.
All assays were performed in triplicate.

[0225] Samples of 20-30 ml blood obtained from healthy donors or BMT
patients, containing 500 units (U) of heparin was added to 50 ml sterile
tubes containing 15 ml Lymphoprep® (Axis shield PoC AS, Oslo Norway).
The blood was added gently without mixing between the Ficoll and the
blood. The tubes were centrifuged for 30 minutes at 1000 g without
brakes. The upper layer that contains the serum was removed and the Buffy
coat that contains the peripheral blood mononuclear cells (PBMCs) was
transferred to new tubes. The PBMCs were washed twice with 40 ml of
phosphate buffer saline (PBS) and 2 mM EDTA (centrifuged at 700 g for 8
minutes). The PBMCs were resuspended in 20 ml PBS, counted, centrifuged
at 500 g for 8 minutes and resuspended in PBMCs medium at
1-5×106 cells/ml. About 70×106 cells are isolated
from a total of 50 ml blood sample.

Example 1

Selection and Cloning of Recombinant Antibodies Specific for HLA-A2-PP65
Complex

[0228] Recombinant peptide-HLA-A2 complexes that present the
pp65495-503 (SEQ ID NO:3) CMV-derived peptide were generated using a
single-chain MHC (scMHC) construct according to the method previously
described previously (Denkberg G., et al., 2000). In this construct, the
extracellular domains of HLA-A2 are connected into a single-chain
molecule with β2m using a 15-aa flexible linker (the β2m
is translationally fused upstream of the MHC heavy chain). The
scMHC-peptide complexes were produced by in vitro refolding of inclusion
bodies in the presence of the pp65 495-503 peptide (SEQ ID NO:3). The
refolded scHLA-A2/pp65 complexes were found to be pure, homogenous, and
monomeric by SDS-PAGE and size exclusion chromatography analyses (data
not shown). Recombinant scMHC-peptide complexes generated by this
strategy were previously characterized in detail for their biochemical,
biophysical, and biological properties, and were found to be correctly
folded and functional (Denkberg G., et al., 2000; Denkberg G., et al.,
2001).

[0229] A large human Fab library containing 3.7×1010 different
Fab clones was used for the selection on biotinylatd HLA-A2/pp65
complexes (De Haard H J., et al., 1999). Phage displayed antibodies which
were capable of binding to the specific biotinylated HLA-A2/peptide
complex were selected as previously described (Denkberg G., et al., 2002;
Lev A., et al., 2002). Enrichment in phage titer was observed after three
rounds of panning (Table 2, hereinbelow). Specificity of the selected
phage antibodies against the complex was analyzed by a differential ELISA
assay in which binding was tested against specific (pp65 495-503 peptide;
SEQ ID NO:3) and non specific (gp100 280-288 peptide; SEQ ID NO:4)
biotinylated HLA-A2/peptide complexes. These were immobilized to wells
through BSA-biotin-streptavidin. As shown in FIG. 1a, a high percentage
of specific clones was observed; 54 clones of the 96 screened (56%), were
peptide specific and bound the specific peptide/MHC used in the selection
(i.e., the scHLA-A2/pp65 complex).

[0230] Cloning of Two Fab Clones with Specificity to the
HLA-A2-pp65495-503 Complex

[0231] The diversity within the selected TCR-like Fabs was assessed by DNA
fingerprint analysis using the BstNI restriction enzyme. The analysis
revealed two different clones, termed H9 and F5 with HLA-A2/pp65
specificity (data not shown). DNA sequencing analysis confirmed these
observations. The nucleic acid and amino acid sequences of the heavy and
light chains of H9Fab clone are provided in FIGS. 14a-d (SEQ ID
NOs:16-19). The nucleic acid and amino acid sequences of the heavy and
light chains of F5 Fab clone are provided in FIGS. 15a-d (SEQ ID
NOs:20-23). The amino acid sequences of the CDRs of the H9 and F5 Fab Abs
are provided in Table 3, hereinbelow. The nucleic acid sequences of the
CDRs of the H9 and F5 Fab Abs are provided in Table 4, hereinbelow.

[0233] The isolated Fab clones with specificity toward the HLA-A2/pp65
complex (H9, F5) were produced in a soluble form in E. coli BL21 cells.
These Fabs which are tagged at the CH1 domain with a hexahistidine
sequence, were purified from the periplasmic fraction by metal affinity
chromatography. SDS-PAGE analysis revealed the level of purification and
the expected molecular size of the Fab antibodies (FIG. 1b).

[0239] To demonstrate that the isolated Fab antibodies can bind the
specific MHC-peptide complex not only in the recombinant soluble form,
but also in the native form, as expressed on the cell surface, the
present inventors used murine TAP2 (transporter associated with antigen
presentation)-deficient RMA-S cells transfected with the human HLA-A2
gene in a single-chain format (Pascolo S., et al., 1997)
(HLA-A2.1/Db-β2m single chain, RMA-S-HHD cells). The
pp65495-503 peptide and control peptides were loaded on RMA-S-HHD
cells and the ability of the selected Fab Abs to bind to peptide-loaded
cells was monitored by flow cytometry. Peptide-induced MHC stabilization
of the TAP2 mutant RMA-S-HHD cells was demonstrated by the reactivity of
mAbs w6/32 (HLA conformation dependent) and BB7.2 (HLA-A2 specific) with
peptide-loaded, but not unloaded cells (data not shown). As shown in
FIGS. 2b and d, Fabs H9 and F5 reacted only with pp65-loaded RMA-S-HHD
cells, but not with cells loaded with the EBV derived peptide. Similar
results were observed in FACS analysis using 10 other HLA-A2-restricted
peptides (data not shown).

[0240] In addition, the present inventors used the TAP.sup.+
EBV-transformed B-lymphoblast HLA-A2.sup.+ JY cells as APCs. These cells
have normal TAP; consequently, peptide loading is facilitated by the
exchange of endogenously derived peptides with HLA-A2-restricted peptides
supplied externally by incubation of the cells with the desired peptides.
As shown in FIGS. 2a and c, the Fab antibodies recognize only JY cells
loaded with the specific pp65 peptide to which they were selected, but
not with control HLA-A2-restricted peptides derived from melanoma gp100
[G9-154 (SEQ ID NO:15) and G9-280 (SEQ ID NO:4) epitopes] and MART1
peptides (SEQ ID NO:11), or a telomerase human telomerase reverse
transcriptase (hTERT)-derived peptide (T540 epitope; SEQ ID NO:6). As a
control, peptide-loaded HLA-A2.sup.-/HLA-A1.sup.+ APD B cells were used.
No binding of the Fab Abs to these cells was observed (data not shown).
These results demonstrate the ability of the selected Fabs to detect
specifically complexes of HLA-A2 in association with the pp65495-503
peptide (SEQ ID NO:3), on the surface of cells.

[0241] These results demonstrate the fine specificity of the recombinant
Fab clones H9 and F5 to soluble or membrane-presented CMV-MHC class I
complex.

Example 3

Generation of Multivalent Antibody Forms and their Binding to
Peptide-Pulsed APCS

[0242] Experimental Results

[0243] Increased Avidity of Fab Tetramers to Peptide Pulsed APCs

[0244] Fab fragments w/o peptide isolated from the phage library are
monovalent. To increase the avidity of these fragments, Fab tetramers
were generated. This approach was previously used to increase the binding
avidity of peptide-MHC complexes to the TCR or to increase the
sensitivity of recombinant Ab molecules (Cloutier S M., et al., 2000). To
form a Fab tetramer with H9, a BirA tag sequence for site-specific
biotinylation was introduced at the C-terminus of the light chain. The
Fab domains were expressed separately in E. coli and were refolded in
vitro followed by purification and in vitro biotinylation using the E.
coli-derived BirA enzyme (Cohen C J., et al., 2002). H9 Fab tetramers
were generated with a fluorescently labeled streptavidin and their
reactivity was examined by flow cytometry with JY pulsed cells. As shown
in FIG. 3a the fluorescence intensity measured on peptide-pulsed JY cells
with the H9 Fab tetramer was significantly higher compared to the
reactivity of the H9 Fab monomer. The specificity, however, was not
altered (FIG. 3c).

[0246] Another strategy for increasing the avidity was by creating a whole
IgG antibody molecule which is bivalent. To transform the recombinant Fab
fragment into a whole IgG molecule, eukaryotic shuttle expression vectors
containing the constant regions of IgG1 for the heavy chain and a vector
containing the constant domain of a kappa light chain were used.
Recombinant H9 Fab-derived IgG was produced from these expression vectors
by co-transfection of the two constructs into human embryonic kidney
HEK293 cells. After proper selection and generation of stable secreting
clones, purified TCR-like whole IgG molecules were produced and tested
for binding specifically towards APCs pulsed with the pp65495-503
peptide. As shown in FIG. 3b, the binding specificity of the whole IgG
molecule was maintained. As expected, the fluorescence intensity observed
with the IgG was significantly higher compared to that of the Fab
monomer. JY cells pulsed with control peptide (derived from gp100) were
incubated with the three H9 constructs (monomer, tetramer, whole IgG Ab)
to confirm specificity (FIG. 3c).

[0247] These results demonstrate the generation of bivalent (IgG) or
tetrameric Fab antibodies and the increased avidity, yet without
compromising specificity of the recombinant antibodies to the CMV-MHC
class I complex.

Example 4

The TCR-Like Antibodies of the Invention are Highly Specific and Sensitive
to MHC-CMV Peptide Complexes

[0248] Experimental Results

[0249] Determination of Binding Affinity of the Recombinant TCR-Like
Antibodies

[0250] Binding affinity determination of the H9 Ab was performed by
surface plasmon resonance (SPR) analysis using streptavidin sensor chips
coated with biotinylated HLA-A2/pp65 or control HLA-A2/EBV complexes. The
apparent affinity of the monomeric/IgG forms of the H9 Ab indicated
KD values of 8 nM and 5 nM, respectively. The time necessary for
binding of the H9 Fab/IgG Ab to the specific complexes (Kon) was
1.05×105 1/Ms and 5.99×105 1/Ms, respectively. The
dissociation rate (Kd or Koff) of the H9 Fab was 8.79×104
1/s compared to the H9 IgG Ab, which was 3.52×10-3 1/s (FIGS.
4a, b). No significant binding of the antibodies was detected when
control HLA-A2/EBV complexes were immobilized to the sensor chip (FIG.
4c).

[0251] The Recombinant TCR-Like Antibodies are Highly Specific to the
MHC-pp65 Complex

[0252] To study the sensitivity of ligand recognition by the Fab and its
derivatives the reactivity threshold was examined by peptide titration on
JY cells which were pulsed with different concentrations of the pp65
495-503 peptide. As shown in FIGS. 5a and b, peptide titration of pulsed
JY demonstrated that the staining intensity was dependent on the
concentration of the peptide used for pulsing, and that peptide
concentrations at the low nM range were sufficient for Fab tetramer (FIG.
5b) but not for the monomer (FIG. 5a). Thus, the tetrameric form of H9
Fab was able to detect much lower numbers of peptide/HLA-A2 complexes on
the surface of peptide-pulsed JY cells than the monomer. Similar results
were observed with the whole IgG molecule (data not shown). Overall,
these and additional studies revealed that the H9 tetramer and IgG
molecules are capable of detecting HLA-A2/pp65 complexes on cells pulsed
with as low as ˜100 nM pp65495-503 peptide.

[0253] The Recombinant TCR-Like Antibodies can Detect Low Amounts of
MHC-pp65 Complexes Presented on Cells in a Mixed Population of Cells

[0254] The TCR-like Fab were further used to detect APCs bearing the
specific peptide-MHC complexes in a heterogeneous cell population. This
can verify the ability of the TCR-like Fab molecules to detect complexes
on individual cell samples in a mixed cell population. To simulate the
situation of a heterogeneous population of cells in which only a small
fraction might express the specific peptide-MHC complex, pp65 peptide
pulsed JY cells were mixed with HLA-A2.sup.-/HLA-A1.sup.+ APD B cells at
various ratios and the reactivity of H9 Fab was analyzed by flow
cytometry. As shown in FIG. 5C, staining with H9 Fab tetramer allows
accurate identification of the admixed pp65 JY pulsed cells that express
on their surface HLA-A2/pp65 complexes, using a simple one-color flow
cytometry analysis. Using various ratios of mixtures between pulsed and
nonpulsed cells, the H9 Fab was shown capable of detecting as low as 5%
pp65 JY pulsed cells within a background population of 95% nonpulsed
cells (FIG. 5C).

The TCR-Like Antibodies of the Invention can Detect HLA-A2/pp65 Complexes
on Surface of Viral-Infected Cells

[0256] Experimental Results

[0257] Detection of HLA-A2/pp65 Complexes on the Surface of Virus-Infected
Cells

[0258] To test the ability of the isolated Fab to bind specifically
HLA-A2/pp65 complexes produced under naturally occurring physiological
Antigen (Ag) processing, HLA-A2 positive fibroblasts were infected with
the CMV laboratory strain AD169 at multiplicity of infection (MOI) of 0.5
(FIGS. 6a-1). HLA-A2 negative fibroblasts infected with the virus, were
used as control in addition to uninfected HLA-A2 negative and positive
cells. 72 hours after infection, infected and control cells were
incubated with the tetrameric form of H9. To verify the expression of
HLA-A2 molecules on the surface of infected, versus uninfected cells, the
human fibroblasts were also stained with PE-labeled BB7.2. Confirmation
for efficiency of virus infection was monitored with anti pp65 mAb and
the secondary antibody FITC-labeled anti mouse IgG. As shown in FIGS. 6a
and c, there was a somewhat decrease in the expression of HLA-A2
complexes on the surface of the virus infected cells, due to the virus
well known down regulation mechanism of the MHC expression. However,
despite the relatively low amount of HLA-A2 expressed on the cell
surface, there was still specific staining of infected cells with the H9
tetramer (FIGS. 6e and g), suggesting that the isolated antibody was able
to detect not only complexes presented on peptide pulsed APCs but also
specific MHC-peptide complexes expressed after active and naturally
occurring endogenous intracellular processing. The H9 Ab showed no
binding at all in the control uninfected cells (FIGS. 6g and h) as well
as in the HLA-A2 negative cells (FIG. 6f), indicating its fine
specificity towards HLA-A2/pp65 complexes presented on the cell surface.
Staining with the anti pp65 mAb revealed the expression of the pp65
protein after successful infection of the fibroblasts (FIGS. 6i and j).

[0259] The specificity of the H9 Ab was verified using a control TCR-like
Ab (2F1) which recognizes specifically class I MHC complexes in
association with the gp100 280-288 peptide. No staining was visible in
this assay, confirming again the H9 tetramer's specificity (data not
shown).

[0260] These results demonstrate, for the first time, the ability to
follow the CMV-MHC class I complexes on the cells surface of APC as well
as inside infected cells.

Example 6

The TCR-Like Antibodies of the Invention can Compete with CTLS on Specific
HLA-A2/pp65 Sites and Thereby Prevent CTL-Mediated Cytotoxicity

[0261] Experimental Results

[0262] The H9 Ab can Prevent CTL-Mediated Cytotoxicity Directed Against
the HLA-A2-pp65 Complex

[0263] The specificity of the H9 Ab to the MHC-pp65 495-503 complex
presented on cells was further demonstrated by the specific inhibition of
CTL-mediated cell killing by the H9 antibody. Briefly, fibroblast cells
were radioactively labeled with S35-methionine before infection with
the CMV virus and 72 hours later the cells were incubated with H9 Ab.
CTLs from a line targeted to the pp65 (495-503) epitope were added at a
target (i.e., fibroblast cells)--effector (i.e., CTL) ratio of 1:10 and
incubated for five hours. Cells incubated with anti-HLA-A2 W6/32 MAb were
used as positive control, while cells without any Ab incubation served as
a reference for maximal killing. As shown in FIG. 6m, maximal percentage
of killing was observed in the virus infected cells which were not
incubated with Abs (CMV CTL alone). However, incubation with the H9 IgG
Ab exhibited ˜60% blockage of killing by the CTLs (CMV CTL+H9).

[0264] The cytotoxicity assay demonstrated the capability of the isolated
antibody to recognize specifically complexes presented on virus infected
cells and its potential to compete with the same sites recognized by
CTLs, leading to the blockage of killing by these effector cells.

Example 7

The TCR-Like Antibodies of the Invention are Valuable Tools for Following
the Dynamics of HLA-A2/pp65 Expression in Cells Infected with the CMV
Virus

[0265] Experimental Results

[0266] The Dynamics of HLA-A2/pp65 Complex Expression in Cells Infected
with Wild-Type and Mutant Virus

[0267] The fact that the H9 Ab was able to detect specific complexes on
virus infected cells enabled to follow the expression levels of the
complexes throughout the virus infection cycle. Based on precedent
results which showed down regulation of MHC class I expression after
viral infection (Ahn, K. et al. 1996), the present inventors investigated
whether the generation and presentation of HLA-A2/pp65 complexes
throughout various time points after infection is influenced by the down
regulation mechanism. To this end two strategies were employed; (i) the
intracellular versus extracellular staining with H9 or anti-HLA-A2 BB7.2
Abs which enabled to determine if the level of the complexes
generation/expression is correlated with their uptake to the cell
surface; (ii) the usage of a mutant strain of CMV which does not induce
down regulation of MHC class I. The level of expression of HLA-A2/pp65
complexes in cells infected with the wild type AD169 strain was compared
to that in cells infected with the mutant strain. For this purpose, the
genetically modified CMV strain RV798 (Jones T R and Sun L., 1997), which
lacks most of the genes responsible for the down regulation mechanism of
MHC class I (US2 to US11 genes), was employed.

[0268] As shown in FIGS. 7a-t, 8a-t and 9a-y, the general expression of
HLA-A2 class I MHC was followed throughout four time points (36, 72, 96
and 120 hours) after cell infection with AD169 WT CMV strain (FIGS. 7a-t)
and RV798 mutant CMV strain (FIGS. 8a-t), as well as the expression of
specific HLA-A2 complexes in association with the pp65 495-503 peptide
using the H9 IgG Ab. The infection efficiency was monitored by following
the expression of the pp65 protein in infected cells through the use of
an anti-pp 65 MAb. Detection with H9 or BB7.2 Abs was performed in each
time point by intracellular and extracellular staining. To verify the
specificity of the reagents used for detection, especially the reactivity
of the anti-HLA-A2/pp65 495-503 TCR-like antibody, controls which were
uninfected HLA-A2 positive fibroblasts (FIGS. 9a-t) or CMV infected human
fibroblasts that are HLA-A2 negative (FIGS. 9u-y) were used. The results
show progressive expression of pp65 in cells infected with wild-type
(FIGS. 7e, j, o, t) and mutant (FIGS. 8e, j, o, t) CMV strains while in
non-infected cells (FIGS. 9e, j, o, t) no expression was observed. The
expression of pp65 in cells that were infected with the mutant stain
RV798 was somewhat higher. Staining with the anti pp65 Ab also indicated
that the cells begin to express the pp65 protein less than 36 hours after
infection (data not shown). These data are in agreement with previous
studies (Soderberg-Naucler C., et al., 1998). Expression of HLA-A2 on the
surface of cells infected with wild-type virus clearly showed a phenotype
involving significant down regulation of HLA-A2 expression (FIGS. 7c, h,
m and r) compared to the uninfected fibroblasts (FIGS. 9c, h, m, r). This
down regulation is increased over time through the progression of the
time points. Also, the intracellular expression of HLA-A2 in infected
cells seemed to be higher than the amount in the uninfected cells
(Compare FIGS. 7d, i, n and s to FIGS. 9d, i, n and s, respectively).
These data are in agreement with previous studies (Ahn K., et al., 1996).

[0269] When cells were infected with wild-type virus, a specific and
gradual increase in staining with the H9 IgG TCR-like antibody was
observed indicating the generation of HLA-A2/pp65 495-503 complexes
inside infected cells (FIGS. 7b, g, l and q) as well as their
presentation on the cell surface (FIGS. 7a, f, k and p). However,
although the amount of complexes which bear the pp65 495-503 peptide
seemed to be quite low at the cell surface (e.g., compare FIG. 7f with
7g), intracellular staining of these specific complexes revealed a very
significant large pool of complexes inside the cell. This might indicate
that although the pp65 is well processed inside the cell and its peptides
are deposited on the class I MHC, it is avoided from being displayed on
the cell surface as part of the virus evasion mechanisms. Interestingly,
there was no correlation between HLA-A2 down regulation as clearly
observed through the progression of time and the significant increase in
the intracellular pools of HLA-A2/pp65 495-503 complexes or their
expression on the cell surface. Most striking is that after 120 hours the
expression of HLA-A2 is very low however both intracellular pools are
very high and surface expression is significant.

[0270] The Reactivity of the H9 IgG Molecule to the MHC-CMV pp65 Peptide
Complex Both Inside and on the Surface of Cells is Highly Specific

[0271] Non-infected HLA-A2 positive cells were stained with anti-HLA-A2
antibody BB7.2 both inside (FIGS. 9d, i, n, s) and on the surface (FIGS.
9c, h, m, r). As shown, there were no observed alterations in HLA-A2
expression inside the cells as well as its presentation on the cell
surface throughout the time points tested after infection. In contrary to
the infected fibroblasts, the amount of complexes as determined using the
BB7.2 antibody on the cell surface of non-infected cells seemed to be
higher than their amount inside the cells (compare FIGS. 9c, h, m, r with
FIGS. 9d, i, n, s, respectively). No pools of complexes were observed
inside the cells (FIGS. 9d, i, n, s) as seen in the infected fibroblasts
(FIGS. 7d, i, n, s). This implies that the HLA-A2 complexes expressed
inside the uninfected cells are freely presented on the cell surface, in
contrast to the infected cells (see FIGS. 7d, i, n, s). In contrast, the
H9 TCR-like antibody was not reactive with uninfected cells both inside
(FIGS. 9b, g, l, q) and on the cell surface (FIGS. 9a, f, k, p),
indicating its fine specificity towards its antigen.

[0272] When HLA-A2 negative human fibroblasts were infected with wild-type
CMV, pp65 expression was clearly observed (FIG. 9y), however, no
reactivity with the anti-HLA-A2 antibody (FIGS. 9w, x) or the H9 TCR-like
antibody (FIGS. 9u, v) was observed inside or on the surface of the
infected cells indicating the highly specific reactivity of the
molecules.

[0273] The presentation of HLA-A2/pp65 complexes was further examined both
inside the cells and on their surface less than 24 hours after infection.
These studies demonstrated that although pp65 is expressed, there is no
presentation of its peptides on HLA-A2 molecules (Data not shown).

[0274] FIGS. 8a-t follow the dynamics of antigen presentation in the
mutant strain RV798. The infected cells were efficiently infected with
the virus as observed from the staining with anti-pp65 (FIGS. 8e, j, o,
t). It was clearly observed that the effect of the mutant virus on HLA-A2
expression inside and on the surface of infected cells was diminished,
thus the mutant virus no longer significantly down regulates HLA-A2
expression, as expected. When using the H9 IgG TCR-like antibody, similar
to the results observed with wild-type CMV, a gradual increase over time
of intracellular pools of HLA-A2/pp65 495-503 complexes inside infected
cells was observed (FIGS. 8b, g, l, q) as well as their gradual
appearance on the cell surface (FIGS. 8a, f, k, p). Also, it was quite
evident that the number of HLA-A2/pp65 495-503 complexes inside the
infected cells was higher than those on the cell surface (Compare FIGS.
8b, g, l, q to FIGS. 8a, f, k, p, respectively). This may indicate that
although the mutant virus does not activate the down regulation
mechanism, there are still HLA-A2 pools as well as specific HLA-A2/pp65
pools inside the cells, which are avoided from being presented on the
cell surface.

[0275] In general, these results present the usage of the H9 Ab to follow
the dynamic expression and kinetics of HLA-A2/pp65 495-503 presentation
intracellularly and on the surface of infected cells as a function of
time after viral infection. Most striking is the observation that there
is no correlation between class I MHC down regulation induced by
wild-type virus and the generation/presentation of the viral specific
HLA-A2/pp65 495-503 complex. On the contrary, the down regulation did not
affect the generation of a significant and large intracellular pool of
viral complexes and their appearance over time on the cell surface.
Similar studies using the H9 antibody and a mutant virus that abolishes
class I MHC down regulation showed a similar pattern of expression inside
the cell and on its surface with somewhat increased number of complexes
on both compared to wild-type virus especially between 24-72 hours after
infection.

Example 8

The TCR-Like Antibodies of the Invention can be Used to Quantify the
Number of HLA-A2/pp65 Complexes on Viral Infected Cells

[0276] The knowledge of the number of complexes presented on the cell
surface can be used to understand how the immune system identifies viral
infection. Related to the studies presented herein, the present inventors
attempted to quantify and compare the number of complexes generated
inside the infected cells to those presented on the cell surface, as
follows.

[0277] Experimental Results

[0278] Quantization of the Number of HLA-A2/pp65 Complexes on the Surface
of Infected Cells

[0279] The unique H9 IgG TCR-like antibody enables the present inventors
to directly quantify the number and percentage of specific HLA-A2/pp65
complexes among HLA-A2-derived complexes which are displayed on the cell
surface. Staining of virus infected cells with the H9 IgG TCR-like
antibody enabled the present inventors to directly count the number of
complexes on the surface of the infected cells using a PE-labeled anti
kappa secondary monoclonal antibody that generates a 1:1 binding
stoichiometry with the H9 IgG molecule. The level of fluorescence
intensity resulting from specific reactivity of the H9 IgG antibody on
infected cells can be directly correlated with the fluorescence
intensities of calibration beads with known numbers PE molecules per bead
(QuantiBRITE PE beads; BD Biosciences), using simple flow cytometry
calibrations. This strategy enabled the present inventors to determine
the number of PE molecules bound to the cells and thereby the number of
sites which are bound by the H9 antibody.

[0280] In agreement to the results presented on FIGS. 7-9 (Example 7,
hereinabove), there was an immediate and massive down regulation of
HLA-A2 complexes (using the BB7 Ab) from the cell surface after infection
with the CMV wild-type strain (FIG. 10d). In all time points there were
about 5,000 complexes observed on the cell surface compared to
˜25,000 complexes in the uninfected cells, implying that there was
over 85% decrease in the amount of HLA-A2 complexes presented on the cell
surface (FIG. 10d). The number of HLA-A2 complexes inside the cells in
infected vs uninfected cells remained almost the same (FIG. 10c). The
number of HLA-A2/pp65 complexes presented on the cell surface was
gradually increased over time (FIG. 10b). Specific complexes were
observed using the H9 antibody starting at 36 hours after infection and
the number reached to approximately 400 sites/cell 120 hours after
infection (FIG. 10c). This implies that 120 hours after infection with
the virus, about 10%-15% of the HLA-A2 complexes presented on the cell
surfaces bear the pp65 495-503 peptide. Interestingly, the number of
these specific complexes inside the cells reaches to ˜2000/cell
after 120 hours (FIG. 10a). This number is close to the total number of
HLA-A2 complexes inside the cell, suggesting that most of the HLA-A2
complexes which accumulate inside infected cells are HLA-A2/pp65. This
might also suggest that most of these specific complexes which are
generated inside the cells are avoided from being presented on the
surface.

[0281] Using the mutant virus, the same number of HLA-A2 molecules on the
cell surface was observed as in the uninfected cells (FIG. 10d). The
number of sites reached to approximately 20,000 (FIG. 10d). However, the
number of complexes quantified inside the cells was significantly higher
than the number observed in the uninfected cells, and approached to
˜10,000 (FIG. 10c) compared to ˜1,000 in the uninfected cells
(FIG. 10c). As for HLA-A2/pp65 complexes, there were ˜400 sites
detected on the cell surface (FIG. 10b), implying that similar to cells
infected with wild-type virus most of viral HLA-A2/pp65 complexes are
avoided from being transported to the cell surface. The percentage of
these complexes amongst HLA-A2 complexes on the cell surface is very low.
However, the number of HLA-A2/pp65 complexes inside the infected cells
reached to approximately 3,000 (FIG. 10a) in each time point after 72
hours thus until 120 hours after infection there is an accumulation of
the specific complexes inside the cell. This accumulation might lead to
the observation that after this time point, most of the complexes inside
the cell are composed of HLA-A2/pp65.

[0282] These data provide a quantitative measure to the observation that
specific HLA-A2/pp65 complexes are being generated in large amounts and
accumulated inside the infected cell in a mechanism that is independent
to the overall down regulation of HLA-A2 molecules in these cells. The
accumulation was observed with wild-type and mutant virus strains and for
both the accumulated HLA-A2/pp65 complexes were avoided from being
presented in large amounts on the cell surface.

[0283] These results visualize large intracellular pools of the viral
complexes after infection, follow and quantify their expression on the
surface. These results demonstrate that despite significant down
regulation of MHC expression by wild-type virus large pools of specific
viral complexes are generated intracellularly, and their export to the
cell surface occurs in a limited quantity. These studies describe the
first attempt to directly visualize and analyze the dynamics of a
naturally occurring viral-derived human MHC-peptide complex after viral
infection.

[0284] The data also demonstrate the ability of the TCR-like antibody of
the instant application to detect and accurately quantify the number of
HLA-A2/peptide complexes on the surface of infected cells under naturally
occurring intracellular processing. These results can be used to follow
the effectiveness of viral strategies for immunization.

[0287] Confocal microscopy of CMV infected cells stained with the H9 IgG
TCR-like antibody enabled the present inventors to visualize and image
the specific HLA-A2/pp65 complexes generated inside the cells, as well as
their display on the cell surface. Moreover, it enabled the present
inventors to localize the complexes inside the cell during the virus
infection cycle.

[0288] CMV infected cells were harvested every 24 hours for 5 days. At
each time point cells were stained with the H9 Ab, and anti human alexa
fluor488 as a secondary Ab. The cells were also stained
intracellularly with the H9 Ab, anti calnexin, cis Golgi matrix protein
(GM130), and anti pp65 Ab, after fixation and permeabilization. Secondary
antibody for the ER marker, Golgi marker and anti pp65 was anti mouse
alexa fluor594. Noninfected fibroblast cells were used as a control.

[0289] The results of these assays further demonstrate and image the
significant pool of specific HLA-A2/pp65 complexes generated inside
infected cells (FIGS. 11a-o, 12a-o). The data also show that the specific
complexes are densely colocalized with the cis-golgi apparatus (FIGS.
11a-o). This co-localization is observed clearly after 24 hours in
comparison with the later time points, in which the complexes are more
widely distributed and co-localized to the ER/cytosol as indicated by
co-staining with the various localization markers (FIGS. 12a-o).
Additionally, as time progresses, a significant enlargement of the Golgi
apparatus is observed, as part of the morphological changes of the
infected cells. Extracellular staining of the HLA-A2/pp65 complexes
showed their display on the cell surface only after 72 hours post
infection (FIGS. 13a-e). These results are with complete agreement with
the flow cytometry analysis of the kinetic of HLA-A2/pp65 epitope
presentation as shown in FIGS. 7a-t, 8a-t and 9a-y. Confocal microscopy
analysis of control noninfected cells showed no staining with the H9 Ab
(FIGS. 13f-h), indicating its fine specificity towards the HLA-A2/pp65
complexes. Staining with anti pp65 Ab confirmed the effectiveness of the
viral infection in the experiments (FIGS. 13i-j).

[0290] These results visualize the present inventors' finding that
specific HLA-A2/pp65 complexes are being generated and accumulate in
infected cells and are localized in the Golgi compartment. They are
prevented from being displayed on the cell surface at early time points
and only 72 hours after infection they can be imaged on the cell surface.
The fact that the specific complexes are prevented from being displayed
on the cell surface is only temporary. Progressed time scales showed that
the complexes are being significantly displayed on the cell surface. The
intermediate time points clearly show that the complexes are less
co-localized with the Golgi due to their movement to the cell membrane.
The phenomena of Golgi enlargement is usually attributed to an extensive
synthesis of proteins after viral infection. These results can imply that
this enlargement is also due to the specific accumulation of complexes in
the Golgi.

[0293] Table 137 hereinbelow, depicts subsequence residue listing
(Sequence), SEQ ID NO: and scoring results [Rank and Score (the estimate
of half time of disassociation of a molecule containing this
subsequence)] obtained according to the user parameters and scoring
information summarized in Tables 5-136, hereinabove, for HLA restricted
peptides derived from the CMV pp65 (SEQ ID NO:53) or pp64 (SEQ ID NO:52)
polypeptides. For each row, a reference to the relevant "user parameters
and scoring information Table" is made by indicating the "Table No." on
the last column.

TABLE-US-00137
Lengthy table referenced here
US20130101594A1-20130425-T00001
Please refer to the end of the specification for access instructions.

Example 12

Detection of HLA-A2/pp65 Complexes on the Surface of Virus-Infected Cells
of Patients

[0294] The ability of the H9 Ab to detect HLA-A2/pp65 complexes was
further evaluated in heterogeneous population of cells taken from CMV
infected individuals. Briefly, samples were taken from bone marrow
transplanted (BMT) patients whom are under reactivation of CMV infection
due to immuno-suppression. Healthy donors were used as a control to
verify the H9 Fab specificity.

[0295] Experimental Results

[0296] Peripheral blood mononuclear cells (PBMCs) were isolated from
samples taken from BMT patients and healthy donors. The isolated cells
were stained with the H9 Ab and the secondary anti human alexa
fluor488 Ab. For intracellular staining with the H9 Ab, the cells
were permeabilized as described under "General Materials and Experimental
Methods".

[0297] Both healthy donors and BMT patients were HLA-A2+ (i.e., express
the HLA-A2 allele) as detected by the anti HLA-A2 Ab (BB7.2) and anti
mouse alexa fluor488 Abs (FIG. 16a and data not shown).
Extracellular staining with the H9 Ab did not detect complexes of the
HLA-A2/pp65 on the surface of infected cells taken from BMT patients or
healthy controls (FIG. 16b and data not shown). However, as shown in
FIGS. 16c and d, intracellular staining with the H9 Ab demonstrated a
significant binding of the antibody to the infected cells from BMT
patients (FIG. 16c) as compared to the control cells taken from healthy
donors (FIG. 16d). These results confirm the ability of the isolated H9
Ab to detect specific HLA-A2/pp65 complexes not only after directed
infection with laboratory strain of the CMV, but also complexes derived
from cells undergoing reactivation of the virus e.g., due to
immuno-suppression.

[0299] The Release of Complexes Accumulation from their Intracellular
Location to the Cell Surface by Proteasome Inhibitor

[0300] The proteasome inhibitor acetyl-leucyl-leucyl-norleucinal (ALLN;
available from CALBIOCHEM Cat. No. 208750) was used in order to
understand the mechanism by which complexes are prevented from reaching
the cell membrane. The effect of the proteasome inhibitor was examined by
FACS analysis, while treating the infected cells with ALLN at three time
scales after infection. At each time scale, the cells were
extracellularly stained with the H9 Ab and anti human alexa-flour488
as a secondary Ab. As shown in FIGS. 17a-i there was a significant effect
of the inhibitor on the presentation of the complexes on the cell
surface. Presence of the inhibitor at each time scale caused an increased
presentation of the complexes on the cell surface compared to untreated
cells. The effect of the increased presentation was more significant at
the lower time scales, and seamed to reach a steady state at 96 hours
post infection. Control uninfected cells showed no staining with the H9
Ab. Thus, incubation with the proteasome inhibitor ALLN increased
presentation of the MHC/pp65 complexes on the cell surface.

SUMMARY

[0301] In this study, the present inventors have demonstrated the
selection of recombinant Fab Abs directed against a human viral T cell
epitope derived from CMV, from a large nonimmune human Ab phage library.
These Abs exhibit an exquisite, very specific, and special binding
pattern: they can bind in a peptide-specific manner only to HLA-A2/pp65
complexes; hence, these are recombinant Abs with T cell Ag receptor-like
specificity. In contrast to the inherent low affinity of TCRs, these
molecules display the high affinity binding characteristics of Abs, in
the nM range, while retaining TCR specificity. The present inventors have
demonstrated here the ability of these Abs to bind specifically to
recombinant class I peptide-MHC complexes, as well as to complexes
presented on the surface of peptide pulsed APCs.

[0302] An important feature of the TCR-like Fab Abs isolated in this study
is their capability to detect TCR ligands at cell surface densities close
to the threshold limit for T cell recognition. The H9
HLA-A2/pp65-specific TCR-like Fab Ab was able to detect in a reproducible
manner as low as 100 sites/cell. Using flow cytometry, it was possible to
use the H9 Fab Ab to detect the specific ligand on cells pulsed with
peptide concentrations similar to those required to activate T cell
hybridoma or CTL lines to cytokine secretion and within a few fold of the
minimal concentration able to sensitize target cells for lysis in a
short-term assay (Porgador A., et al., 1997).

[0303] These data indicate that when applied to dissociated cell
populations using flow cytometry, the detection of ligand with H9 and
other TCR-like Fabs with similar affinity approaches the sensitivity of T
cells, and hence that these molecules are suitable reagents for
evaluating antigenic complex expression at low, but physiologically
relevant levels. In this study, the detection sensitivity of specific
ligand was observed with as low as 100 complexes per cell. Thus, this
principle has been applied in this study to mixtures of peptide pulsed
HLA-A2+ JY cells, and the HLA-A2-B cell line APD. By using the H9
tetramer in a single-step staining for flow cytometry, it was possible to
readily identify pp65 495-503 peptide pulsed JY cells admixed with APD
cells in as low proportion as 5%.

[0304] The avidity of the TCR-like Ab molecules was improved by making the
recombinant monovalent molecules into multivalent molecules. This was
feasible by altering the basic Fab form to a tetrameric molecule or to a
whole bivalent IgG Ab.

[0305] Detection of class I MHC complexes in association with the pp65
495-503 peptide on virus infected cells, showed the ability of the H9 Ab
to recognize complexes not only on the surface of peptide pulsed APCs,
but also complexes which were produced by naturally occurring active
antigen processing. Cytotoxicity assays directed to virus infected cells
confirmed these findings. The blockage of killing by the CTLs after
incubation with the H9 Ab showed a competition between the cytotoxic
T-cell receptor and the H9 TCR-like Ab on the same site presented on the
virus infected cell.

[0306] Using the H9 Ab at various time points following infection the
present inventors could track the presentation level of HLA-A2/pp65
complexes during the course of virus infection cycle. Specific staining
with the H9 Ab lead to the observation that the expression level of the
specific HLA-A2/pp65 complexes on the cell surface does not represent the
overall quantity of these specific complexes, because as shown most of
them are located inside the cell. The results presented herein
demonstrate the existence of a significant large pool of specific
HLA-A2/pp65 complexes inside virus infected cells, which increased as a
function of time after viral infection. The use of a CMV mutant strain
which lacks the genes responsible for MHC class I down regulation
revealed similar findings. Large pools of specific complexes, bearing the
pp65 495-503 peptide, were found inside the cells. In contrast to the
uninfected cells, there is a large amount of MHC class I complexes inside
the cells which are infected with the wild-type/mutant strain.

[0307] The results of the kinetic assays also clearly show that there is a
great correlation between the pp65 expression level and its presentation
level. Both increase as time goes by. Moreover, the timing of the pp65
expression might precede the processing and presentation of this protein,
as presented in the results.

[0308] This work provides also quantitative data about the number of
specific HLA-A2/pp65 complexes generated inside infected cells as well as
presented on the cell surface after active intracellular processing by
virus infected cells. The results revealed for the first time the number
of sites which are presented on the cell surface and recognized by the
immune system. Moreover, quantization of general HLA-A2 complexes enabled
the present inventors to determine the percentage of complexes down
regulated after viral infection. It also enabled the present inventors to
compare between the number of general complexes and the number of
specific HLA-A2/pp65 complexes inside the cells and on their surface.
This analysis enables the determination of the percentage of the specific
complexes among the general complexes. The results indicated
quantitatively that most of the complexes inside the virus infected cells
are bearing the pp65 495-503 peptide. Large numbers of specific complexes
were also found in the cells infected with the mutant strain,
strengthening the previous data, regarding the pools which are prevented
from being translocated to the membrane.

[0309] Confocal immunofluorescence microscopy enabled for the first time
direct visualization of the intracellular and extracellular sites of
peptide-MHC molecules throughout virus infection cycle, as well as
determination of their localization inside the cell. This visualization
revealed the colocalization of the HLA-A2/pp65 complexes with the
cis-golgi apparatus. It also showed the exact movement of the complexes
from this location to the cell surface, in correlation to the virus
infection kinetics. At the progressed time scales there was a significant
display of the complexes on the cell surface.

[0310] The study presented here shows the usage of an isolated human
recombinant Ab towards a specific viral peptide-MHC class I in the
following: (i) tracking the level of specific complexes throughout time
scale which represents a viral infection cycle; (ii) tracking the number
of complexes throughout time scale inside the cell and on its surface and
analysis of this data; (iii) visualization of complexes in a viral
infection system which demonstrate the intracellular localization of the
complexes throughout time scale, and; (iv) detection of the correlation
between protein expression and its derived peptide presentation on HLA-A2
complexes after processing.

[0311] It is appreciated that certain features of the invention, which
are, for clarity, described in the context of separate embodiments, may
also be provided in combination in a single embodiment. Conversely,
various features of the invention, which are, for brevity, described in
the context of a single embodiment, may also be provided separately or in
any suitable subcombination.

[0312] Although the invention has been described in conjunction with
specific embodiments thereof, it is evident that many alternatives,
modifications and variations will be apparent to those skilled in the
art. Accordingly, it is intended to embrace all such alternatives,
modifications and variations that fall within the spirit and broad scope
of the appended claims. All publications, patents and patent applications
mentioned in this specification are herein incorporated in their entirety
by reference into the specification, to the same extent as if each
individual publication, patent or patent application was specifically and
individually indicated to be incorporated herein by reference. In
addition, citation or identification of any reference in this application
shall not be construed as an admission that such reference is available
as prior art to the present invention.

[0328] LENGTHY TABLES
The patent application contains a lengthy table section. A copy of the
table is available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20130101594A1).
An electronic copy of the table will also be available from the USPTO
upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).